JP6910078B2 - WNT composition and purification method - Google Patents

Description

Background of the Invention Wnt protein is a highly conserved secretory signaling molecule, a family of signaling molecules that bind to cell surface receptors encoded by Frizzled and low density lipoprotein receptor-related proteins (LRPs). To form. The WNT gene family consists of structurally related genes that encode secretory signaling proteins. These proteins are involved in multiple developmental processes, including tumorigenesis, as well as regulation of cell fate and patterning during embryogenesis. Upon binding, the ligand induces a cascade of intracellular events that ultimately results in transcription of the target gene through the nuclear activity of β-catenin and the DNA-binding protein TCF (CleversH). , 2004, Wnt signaling: Ig-norrin the dogma, Curr Biol, Vol. 14: R436-R437; Nelson & Nusse, 2004, Convergence of Wnt, beta-catenin, and cadherin pathways, Science, Vol. 303: 1483-1487. Gordon & Nusse, 200
6 years, Wnt signaling: Multiple pathways, multiple receptors, and multipletranscription factors, J Biol Chem, Vol. 281: pp. 22429-22433).
Wnt proteins are also involved in a wide variety of cell decisions associated with bone development programs. For example, the Wnt protein regulates the expression level of sox9, which affects the commitment of mesenchymal progenitor cells to the fate of skeletal formation. The Wnt protein affects the differentiation of cells into osteoblasts or chondrocytes. In adults, there is evidence that Wnt signaling regulates bone mass. For example, mutations within the human Wnt co-receptor LRP5 include multiple high bone mass syndromes, including type I osteoporosis and endometrial hyperostosis or autosomal dominant osteosclerosis, as well as low bone mass syndromes. It is also associated with osteoporosis-pseudoglioloma, a bone mass disorder. Increased production of the Wnt inhibitor Dkkl is associated with multiple myeloma, a disease that increases bone resorption, as one of its prominent features. For more details, see S. Minear et al., Wnt proteins promote bone regeneration, Sci. Transl. Med., Volume 2, pp. 29-30 (2010); and Zhao et al., Controlling the in vivo activity of Wnt liposomes, Methods Enzymol, See Volume 465: pp. 331-47 (2009).

Clevers H, 2004, Wnt signaling: Ig-norrin the dogma, Curr Biol, Vol. 14, pp. R436-R437 Nelson & Nusse, 2004, Convergence of Wnt, beta-catenin, and cadherin pathways, Science, Vol. 303: 1483-1487 Gordon & Nusse, 2006, Wnt signaling: Multiple pathways, multiple receptors, and multiple transcription factors, J Biol Chem, 281: pp. 22829-22433 S. Minear et al., Wnt proteins promote bone regeneration, Sci. Transl. Med., Volume 2, pp. 29-30 (2010) Zhao et al., Controlling the in vivo activity of Wnt liposomes, MethodsEnzymol, Vol. 465: pp. 331-47 (2009)

Outline of the Invention In a specific embodiment, the functionally active Wnt3a polypeptide, which is a composition containing a functionally active Wnt3a polypeptide and an aqueous solution containing liposomes, is the functionally active Wnt3a polypeptide. A composition comprising a lipid modification at the amino acid position corresponding to the amino acid residue 209 shown in 1 is disclosed. In some embodiments, the functionally active Wnt3a polypeptide is not lipid-modified at the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. In some embodiments, the functionally active Wnt3a polypeptide is integrated into the liposome membrane. In some embodiments, the functionally active Wnt3a polypeptide projects from the liposome membrane to the outer surface of the lipid membrane. In some embodiments, the functionally active Wnt3a polypeptide is not incorporated into the aqueous core of the liposome. In some embodiments, the functionally active Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70 relative to the amino acid sequence set forth in SEQ ID NO: 1. Includes%, about 80%, about 90%, or about 95% sequence identity. In some embodiments, the functionally active Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the concentration of the functionally active Wnt3a polypeptide is between about 5 μg / μL to about 15 μg / μL, or about 8 μg / μL to about 12 μg / μL. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C, about 15 ° C to about 25 ° C, or about 20 ° C to about 25 ° C. In some embodiments, the liposomes have a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. Have. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the Wnt3a polypeptide is a mammalian Wnt3a polypeptide. In some embodiments, the mammal is a human. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt3a polypeptide is further glycosylated. In some embodiments, the composition is a stable composition. In some embodiments, the composition is stable for up to about 106 days without substantial loss of activity. In some embodiments, the composition is stable at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the composition is stable under nitrogen.

As used herein, in a particular embodiment, the composition comprises a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, wherein the phospholipids constituting the liposomes are from about 10 ° C. to about 25 ° C. Compositions having a phase transition temperature of ° C. are disclosed. Also herein, in certain embodiments, a composition comprising a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, the length of the tail carbon of the phospholipids that make up the liposomes. Disclosed are compositions in which is between about 12 carbons and about 14 carbons. In some embodiments, the Wnt polypeptide is integrated into the liposome membrane. In some embodiments, the Wnt polypeptide projects from the liposome membrane to the outer surface of the lipid membrane. In some embodiments, the Wnt polypeptide is not integrated into the aqueous core of the liposome. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide. In some embodiments, the concentration of said Wnt polypeptide is between about 5 μg / μL and about 15 μg / μL, or about 8 μg / μL and about 12 μg / μL. In some embodiments, the liposome has a net charge of 0 between pH about 6.5 and about 8.0. In some embodiments, the liposome has a net positive charge or a net negative charge between pH about 6.5 and about 8.0. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the mammal is a human. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt polypeptide is further glycosylated. In some embodiments, the composition is a stable composition. In some embodiments, the composition is stable for up to about 106 days without substantial loss of activity. In some embodiments, the composition is stable at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the composition is stable under nitrogen.

Here, in a particular embodiment, a method of preparing a liposome Wnt3a polypeptide, wherein (a) the Wnt3a polypeptide is harvested from an acclimatized medium containing mammalian cells; (b) said Wnt3a poly. A step of introducing the peptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized; and (c) a step of eluting the Wnt3a polypeptide from the ion exchange column using a step gradient. (D) A method comprising contacting the Wnt3a polypeptide from step (c) with an aqueous solution of the liposome is disclosed. In some embodiments, the contacting step is performed at a temperature between about 21 ° C and about 25 ° C. In some embodiments, the contact time is between about 30 minutes and about 24 hours. In some embodiments, the step gradient includes a first gradient and a second gradient. In some embodiments, the first gradient contains a buffer solution containing 50 mM potassium chloride, and the second gradient contains a buffer containing about 150 mM potassium chloride to about 1.5 M potassium chloride. Contains solution. In some embodiments, the buffer solution further comprises a detergent. In some embodiments, the surfactant is CH.
APS or Triton X-100. In some embodiments, the conditioned medium comprises up to 10% fetal bovine serum. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the yield of said Wnt3a polypeptide after step (c) is between about 60% and about 90%. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C, about 15 ° C to about 25 ° C, or about 20 ° C to about 25 ° C. In some embodiments, the liposomes have a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. Have. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt3a polypeptide is a mammalian Wnt3a polypeptide. In some embodiments, the mammal is a human. In some embodiments, the conditioned medium comprises serum or serum substitute. In some embodiments, the conditioned medium comprises serum. In some embodiments, the serum is fetal bovine serum. In some embodiments, the serum concentration is between about 0.1% and about 15% in the conditioned medium. In some embodiments, the conditioned medium comprises a serum substitute. In some embodiments, the serum substitute is a lipid-based substitute. In some embodiments, the conditioned medium is a serum-free medium. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt3a polypeptide is further glycosylated. In some embodiments, the Wnt3a product is separated from the sample mixture by centrifugation. In some embodiments, the centrifugation time is up to 1 hour at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the Wnt3a product after centrifugation is resuspended in sterile 1x phosphate buffered saline (PBS). In some embodiments, the Wnt3a product is stable under nitrogen. In some embodiments, the Wnt3a product is stable at temperatures between about 1 ° C and about 8 ° C.

In the present specification, in a specific embodiment, a method for preparing a liposome Wnt polypeptide, which comprises contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, wherein the phospholipid constituting the liposome is about. Methods having a phase transition temperature of 10 ° C to about 25 ° C are disclosed. Further, in the present specification, in a specific embodiment, a method for preparing a liposome Wnt polypeptide includes a step of contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, and the tail of the phospholipid constituting the liposome. Disclosed are methods in which the length of carbon is between about 12 carbons and about 14 carbons. In some embodiments, the contacting step is performed at a temperature between about 21 ° C and about 25 ° C. In some embodiments, the contact time is between about 6 hours and about 24 hours. In some embodiments, the method further comprises a further purification step prior to the step of contacting the Wnt polypeptide with an aqueous solution of liposomes. In some embodiments, the additional purification step is an ion exchange purification step, a hydrophobic purification step, or an affinity purification step. In some embodiments, the further purification step is an ion exchange purification step. In some embodiments, the ion exchange purification step comprises contacting the sample with beads on which a sulfonated polycyclic aromatic compound is immobilized or a column on which a sulfonated polycyclic aromatic compound is immobilized. In some embodiments, the ion exchange purification step comprises a step gradient. In some embodiments, the ion exchange purification buffer comprises a surfactant. In some embodiments, the surfactant is CHAPS or Triton X-100. In some embodiments, the further purification step is a hydrophobic purification step. In some embodiments, the hydrophobic purification step comprises beads with protein A immobilized or a protein A column. In some embodiments, the yield of the Wnt polypeptide after the further purification step is between about 60% and about 99%. In some embodiments, the method further comprises the step of collecting the Wnt polypeptide from a conditioned medium containing cells derived from an expressing cell line. In some embodiments, the expression cell line is a mammalian expression cell line. In some embodiments, the mammalian expression cell line is a Chinese hamster ovary (CHO) cell line. In some embodiments, the conditioned medium comprises serum or serum substitute. In some embodiments, the serum is fetal bovine serum. In some embodiments, the serum concentration is between about 0.1% and about 15% in the conditioned medium. In some embodiments, the conditioned medium comprises a serum substitute. In some embodiments, the serum substitute is a lipid-based substitute. In some embodiments, the conditioned medium is a serum-free medium. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide. In some embodiments, the liposomes have a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. Have. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the Wnt polypeptide is a mammalian Wnt polypeptide. In some embodiments, the mammal is a human. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt polypeptide is further glycosylated. In some embodiments, the Wnt3a product is separated from the sample mixture by centrifugation. In some embodiments, the centrifugation time is up to 1 hour at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the Wnt product after centrifugation is resuspended in sterile 1x PBS. In some embodiments, the Wnt product is stable under nitrogen. In some embodiments, the Wnt3a product is stable at temperatures between about 1 ° C and about 8 ° C.

As used herein, in certain embodiments, the composition of isolated enhanced mammalian bone marrow cells, after treatment with liposome Wnt polypeptide, the cells are Runx2, Osterix, osteocalcin, osteopontin, alkali. A composition having one or more enhanced expression levels of a biomarker selected from the group consisting of phosphatase, type I collagen, Axin2, Lef1, and Tcf4 is disclosed. In some embodiments, the mammalian bone marrow cell is a rodent bone marrow cell. In some embodiments, the rodent bone marrow cells are harvested from rodents older than 10 months. In some embodiments, the mammalian bone marrow cell is a human bone marrow cell. In some embodiments, the human bone marrow cells are obtained from a 35 year old or older subject. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression levels are compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a human subject younger than 35 years. In some embodiments, the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide.

As used herein, in certain embodiments, a composition of mammalian bone marrow cells obtained from a human subject 35 years or older, said said mammalian bone marrow after treatment with a liposome Wnt polypeptide. A composition in which cells express one or more of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. The thing is disclosed. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression levels are compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a human subject younger than 35 years. In some embodiments, the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide.

As used herein, in certain embodiments, it is a mammalian bone marrow composition made by a process comprising contacting an isolated mammalian bone marrow cell ex vivo with a liposome Wnt polypeptide. Mammalian bone marrow compositions are disclosed in which the contact time is between about 30 minutes and about 4 hours. In some embodiments, the contact temperature is between about 0 ° C. and about 37 ° C., or between about 20 ° C. and about 25 ° C. In some embodiments, the process further comprises a washing step of removing the free liposome Wnt polypeptide after the contacting step. In some embodiments, the process further comprises the step of implanting the liposome Wnt polypeptide into a bone defect site. In some embodiments, the bone marrow cells are selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, AXIN2, Left1, and Tcf4 as compared to untreated mammalian bone marrow cells. Has one or more enhanced expression levels of the biomarkers. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the isolated mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is a rodent bone marrow cell. In some embodiments, the rodent bone marrow cells are harvested from rodents older than 10 months. In some embodiments, the mammalian bone marrow cell is a human bone marrow cell. In some embodiments, the human bone marrow cells are harvested from a 35 year old or older subject. In some embodiments, the human bone marrow cells after treatment with the liposome Wnt polypeptide exhibit the biomarker expression levels observed in subjects younger than 35 years. In some embodiments, the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide.

Here, in a particular embodiment, a method of treating a bone defect in a subject, the step of (a) contacting a sample containing mammalian bone marrow cells ex vivo with the liposome Wnt3a polypeptide; A method is disclosed that includes b) washing the sample to remove the free liposome Wnt3a polypeptide; c) implanting the bone marrow graft material treated with the liposome Wnt3a polypeptide into the bone defect site. NS. In some embodiments, the contact temperature is between about 0 ° C. and about 37 ° C., or between about 20 ° C. and about 25 ° C. In some embodiments, the contact time is between about 30 minutes and about 4 hours. In some embodiments, the bone marrow cells are selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, AXIN2, Left1, and Tcf4 as compared to untreated mammalian bone marrow cells. Has one or more enhanced expression levels of the biomarkers. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt3a polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the isolated mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is a rodent bone marrow cell. In some embodiments, the rodent bone marrow cells are harvested from rodents older than 10 months. In some embodiments, the mammalian bone marrow cell is a human bone marrow cell. In some embodiments, the human bone marrow cells are harvested from a 35 year old or older subject. In some embodiments, the human bone marrow cells after treatment with the liposome Wnt3a polypeptide exhibit the biomarker expression levels observed in subjects younger than 35 years. In some embodiments, the liposome Wnt3a polypeptide comprises a Wnt3a polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in.

Here, in a particular embodiment, a method of treating a bone defect in a subject, the step of (a) contacting a sample containing mammalian bone marrow cells ex vivo with a liposome Wnt polypeptide; A method comprising (b) washing the sample to remove the free liposome Wnt polypeptide; (c) implanting the bone graft material treated with the liposome Wnt polypeptide into the bone defect site. It will be disclosed. In some embodiments, the contact temperature is between about 0 ° C. and about 37 ° C., or between about 20 ° C. and about 25 ° C. In some embodiments, the contact time is between about 30 minutes and about 4 hours. In some embodiments, the bone marrow cells are selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, AXIN2, Left1, and Tcf4 as compared to untreated mammalian bone marrow cells. Has one or more enhanced expression levels of the biomarkers. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the isolated mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is a rodent bone marrow cell. In some embodiments, the rodent bone marrow cells are harvested from rodents older than 10 months. In some embodiments, the mammalian bone marrow cell is a human bone marrow cell. In some embodiments, the human bone marrow cells are harvested from a 35 year old or older subject. In some embodiments, the human bone marrow cells after treatment with the liposome Wnt polypeptide exhibit the biomarker expression levels observed in subjects younger than 35 years. In some embodiments, the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide.

In a particular embodiment, the present specification discloses a kit for making a bone graft material containing the liposome Wnt polypeptide. In some embodiments, the liposome Wnt polypeptide is a composition comprising a functionally active mammalian Wnt polypeptide and an aqueous solution containing the liposome, wherein the phospholipids constituting the liposome. It has a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the liposome Wnt polypeptide is a composition comprising a functionally active mammalian Wnt polypeptide and an aqueous solution containing the liposome, wherein the tail of the phospholipid constituting the liposome. The length of carbon is between about 12 carbons and about 14 carbons. In some embodiments, the Wnt polypeptide is incorporated into the liposome membrane. In some embodiments, the Wnt polypeptide projects from the liposome membrane to the outer surface of the lipid membrane. In some embodiments, the Wnt polypeptide is not integrated into the aqueous core of the liposome. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide. In some embodiments, the concentration of said Wnt polypeptide is between about 5 μg / μL and about 15 μg / μL, or about 8 μg / μL and about 12 μg / μL. In some embodiments, the liposome has a net charge of 0 between a pH of about 6.5 to about 8.0. In some embodiments, the liposome has a net positive charge or a net negative charge between pH about 6.5 and about 8.0. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the mammal is a human. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt polypeptide is further glycosylated. In some embodiments, the composition is a stable composition. In some embodiments, the composition is stable for up to about 106 days without substantial loss of activity. In some embodiments, the composition is stable at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the composition is stable under nitrogen.

As used herein, in certain embodiments, a kit for making a bone graft material comprising a liposome Wnt3a polypeptide is disclosed. In some embodiments, the liposome Wnt3a polypeptide is a composition comprising a functionally active mammalian Wnt3a polypeptide and an aqueous solution comprising the liposome, wherein the functionally active Wnt3a polypeptide. Includes a lipid modification at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1. In some embodiments, the functionally active Wnt3a polypeptide is not lipid-modified at the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. In some embodiments, the functionally active Wnt3a polypeptide is incorporated into the liposome membrane. In some embodiments, the functionally active Wnt3a polypeptide projects from the liposome membrane to the outer surface of the lipid membrane. In some embodiments, the functionally active Wnt3a polypeptide is not integrated into the aqueous core of the liposome. In some embodiments, the functionally active Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70 relative to the amino acid sequence set forth in SEQ ID NO: 1. Includes%, about 80%, about 90%, or about 95% sequence identity. In some embodiments, the functionally active Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the concentration of the functionally active Wnt3a polypeptide is between about 5 μg / μL to about 15 μg / μL, or about 8 μg / μL to about 12 μg / μL. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C, about 15 ° C to about 25 ° C, or about 20 ° C to about 25 ° C. In some embodiments, the liposomes have a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. Have. In some embodiments, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome further comprises cholesterol. In some embodiments, the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0. In some embodiments, the Wnt3a polypeptide is a mammalian Wnt3a polypeptide. In some embodiments, the mammal is a human. In some embodiments, the lipid modification is palmitoylation. In some embodiments, the Wnt3a polypeptide is further glycosylated. In some embodiments, the composition is a stable composition. In some embodiments, the composition is stable for up to about 106 days without substantial loss of activity. In some embodiments, the composition is stable at temperatures between about 1 ° C and about 8 ° C. In some embodiments, the composition is stable under nitrogen.

As used herein, in certain embodiments, a kit for making a bone graft material comprising a composition of isolated enhanced mammalian bone marrow cells is disclosed. In some embodiments, the isolated enhanced mammalian bone marrow cell composition is a mammalian bone marrow cell composition obtained from a 35 year old or older human subject, wherein the liposome. After treatment with the Wnt polypeptide, the mammalian bone marrow cells were selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Left1, and Tcf4 at enhanced expression levels. Express one or more of the biomarkers. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the enhanced expression levels are compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. In some embodiments, after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. In some embodiments, the reduced expression levels within the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some embodiments, the bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells. In some embodiments, the reduction in apoptosis levels is about 50%. In some embodiments, the mammalian bone marrow cells comprise enhanced osteogenic potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhanced bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some embodiments, the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells. In some embodiments, said new bone growth levels of transplanted treated mammalian bone marrow cells are about 1% to about 20 compared to new bone growth levels of transplanted untreated mammalian bone marrow cells. % Or between about 5% and about 12%. In some embodiments, the mammalian bone marrow cells include adherent and non-adherent bone marrow cells. In some embodiments, the mammalian bone marrow cells are adherent bone marrow cells. In some embodiments, the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the attached bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an autologous bone marrow cell. In some embodiments, the autologous bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the autologous bone marrow cells are adherent bone marrow cells. In some embodiments, the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the autologous bone marrow cells include bone marrow stromal cells. In some embodiments, the mammalian bone marrow cell is an allogeneic bone marrow cell. In some embodiments, the allogeneic bone marrow cells include attached and non-attached bone marrow cells. In some embodiments, the allogeneic bone marrow cells are adherent bone marrow cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some embodiments, the allogeneic bone marrow cells include bone marrow stromal cells. In some embodiments, the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a human subject younger than 35 years. In some embodiments, the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome. In some embodiments, the phospholipids that make up the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. In some embodiments, the length of the tail carbon of the phospholipids that make up the liposome is between about 12 and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. Contains 90%, or about 95% sequence identity. In some embodiments, the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1 and the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1. Not lipid modified in. In some embodiments, the Wnt polypeptide is a Wnt5a or Wnt10b polypeptide.
The present invention provides, for example, the following items.
(Item 1)
A composition comprising a functionally active Wnt3a polypeptide and an aqueous solution containing liposomes, wherein the functionally active Wnt3a polypeptide is located at the amino acid position corresponding to amino acid residue 209 set forth in SEQ ID NO: 1. A composition comprising a lipid modification.
(Item 2)
The stable composition of item 1, wherein the functionally active Wnt3a polypeptide is not lipid-modified at the amino acid position corresponding to amino acid residue 77 set forth in SEQ ID NO: 1.
(Item 3)
The composition according to item 1 or 2, wherein the functionally active Wnt3a polypeptide is incorporated into a liposome membrane.
(Item 4)
The composition according to any one of items 1 to 3, wherein the functionally active Wnt3a polypeptide projects from the liposome membrane to the outer surface of the lipid membrane.
(Item 5)
The composition according to any one of items 1 to 4, wherein the functionally active Wnt3a polypeptide is not incorporated into the aqueous core of the liposome.
(Item 6)
The functionally active Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about the amino acid sequence set forth in SEQ ID NO: 1. The composition according to any one of items 1 to 5, which comprises 90%, or about 95%, sequence identity.
(Item 7)
The composition according to any one of items 1 to 5, wherein the functionally active Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 8)
The item according to any one of items 1 to 7, wherein the concentration of the functionally active Wnt3a polypeptide is between about 5 μg / μL and about 15 μg / μL, or about 8 μg / μL and about 12 μg / μL. Composition.
(Item 9)
The composition according to any one of items 1 to 8, wherein the length of the tail carbon of the phospholipid constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 10)
Item 2. The composition described.
(Item 11)
Items 1 to 10, wherein the liposome has a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. The composition according to any one item.
(Item 12)
The composition according to any one of items 1 to 11, wherein the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC).
(Item 13)
The composition according to any one of items 1 to 12, wherein the liposome further contains cholesterol.
(Item 14)
The composition according to item 12 or 13, wherein the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0.
(Item 15)
The composition according to any one of items 1 to 14, wherein the Wnt3a polypeptide is a mammalian Wnt3a polypeptide.
(Item 16)
The composition according to item 15, wherein the mammal is a human.
(Item 17)
The composition according to any one of items 1 to 16, wherein the lipid modification is palmitoylation.
(Item 18)
The composition according to any one of items 1 to 17, wherein the Wnt3a polypeptide is further glycosylated.
(Item 19)
The composition according to any one of items 1 to 18, which is a stable composition.
(Item 20)
The composition according to any one of items 1 to 19, which is stable for up to about 106 days without substantial loss of activity.
(Item 21)
The composition according to any one of items 1 to 20, which is stable at a temperature between about 1 ° C. and about 8 ° C.
(Item 22)
The composition according to any one of items 1 to 21, which is stable under nitrogen.
(Item 23)
A composition comprising a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, wherein the phospholipids constituting the liposomes have a phase transition temperature of about 10 ° C to about 25 ° C.
(Item 24)
A composition comprising a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, wherein the tail carbon length of the phospholipids constituting the liposome is between about 12 carbons and about 14 carbons. The composition that is.
(Item 25)
The composition according to item 23 or 24, wherein the Wnt polypeptide is incorporated into a liposome membrane.
(Item 26)
The composition according to any one of items 23 to 25, wherein the Wnt polypeptide projects from the liposome membrane to the outer surface of the lipid membrane.
(Item 27)
The composition according to any one of items 23 to 26, wherein the Wnt polypeptide is not incorporated into the aqueous core of the liposome.
(Item 28)
The composition according to any one of items 23 to 27, wherein the Wnt polypeptide is a Wnt3a polypeptide.
(Item 29)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 28. The composition of item 28, comprising sequence identity of.
(Item 30)
28. The composition of item 28, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 31)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The composition according to any one of items 28 to 30.
(Item 32)
The composition according to any one of items 23 to 27, wherein the Wnt polypeptide is a Wnt5a polypeptide or a Wnt10b polypeptide.
(Item 33)
The composition according to any one of items 23 to 32, wherein the concentration of the Wnt polypeptide is between about 5 μg / μL and about 15 μg / μL, or about 8 μg / μL and about 12 μg / μL.
(Item 34)
The composition according to any one of items 23 to 33, wherein the liposome has a net charge of 0 between a pH of about 6.5 to about 8.0.
(Item 35)
The composition according to any one of items 23 to 34, wherein the liposome has a net positive charge or a net negative charge between about pH 6.5 and about 8.0.
(Item 36)
The composition according to any one of items 23 to 34, wherein the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC).
(Item 37)
The composition according to any one of items 23 to 36, wherein the liposome further comprises cholesterol.
(Item 38)
36 or 37. The composition according to item 36 or 37, wherein the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0.
(Item 39)
The composition according to any one of items 23 to 38, wherein the mammal is a human.
(Item 40)
The composition according to any one of items 23 to 39, wherein the lipid modification is palmitoylation.
(Item 41)
The composition according to any one of items 23 to 40, wherein the Wnt polypeptide is further glycosylated.
(Item 42)
The composition according to any one of items 23 to 41, which is a stable composition.
(Item 43)
The composition according to any one of items 23 to 42, which is stable for up to about 106 days without substantial loss of activity.
(Item 44)
The composition according to any one of items 23 to 43, which is stable at a temperature between about 1 ° C. and about 8 ° C.
(Item 45)
The composition according to any one of items 23 to 44, which is stable under nitrogen.
(Item 46)
A method for preparing a liposome Wnt3a polypeptide, which is a method for preparing a liposome Wnt3a polypeptide.
a) With the step of collecting the Wnt3a polypeptide from a conditioned medium containing mammalian cells;
b) The step of introducing the Wnt3a polypeptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized;
c) With the step of eluting the Wnt3a polypeptide from the ion exchange column using a step gradient;
d) A method comprising contacting the Wnt3a polypeptide from step (c) with an aqueous solution of liposomes.
(Item 47)
46. The method of item 46, wherein the contacting step is performed at a temperature between about 21 ° C and about 25 ° C.
(Item 48)
46. The method of item 46, wherein the contact time is between about 30 minutes and about 24 hours.
(Item 49)
46. The method of item 46, wherein the step gradient comprises a first gradient and a second gradient.
(Item 50)
Item 49, wherein the first gradient contains a buffer solution containing 50 mM potassium chloride, and the second gradient contains a buffer solution containing potassium chloride between about 150 mM potassium chloride and about 1.5 M. The method described.
(Item 51)
The method of item 50, wherein the buffer solution further comprises a surfactant.
(Item 52)
51. The method of item 51, wherein the surfactant is CHAPS or Triton X-100.
(Item 53)
46. The method of item 46, wherein the conditioned medium contains up to 10% fetal bovine serum.
(Item 54)
46. The method of item 46, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
(Item 55)
46. The method of item 46, wherein the yield of the Wnt3a polypeptide after step (c) is between about 60% and about 90%.
(Item 56)
46. The method of item 46, wherein the length of the tail carbon of the phospholipids constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 57)
46 or 56. The method of item 46 or 56, wherein the phospholipids constituting the liposome have a phase transition temperature of about 10 ° C to about 25 ° C, about 15 ° C to about 25 ° C, or about 20 ° C to about 25 ° C.
(Item 58)
Items 46, 56 or, wherein the liposome has a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. The method according to any one of 57.
(Item 59)
The method of any one of items 46 or 56-58, wherein the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC).
(Item 60)
The method of any one of items 46 or 56-59, wherein the liposome further comprises cholesterol.
(Item 61)
59. The method of item 59 or 60, wherein the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0.
(Item 62)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 46. The method of item 46, comprising sequence identity of.
(Item 63)
46. The method of item 46, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 64)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The method according to any one of items 46, 62, or 63.
(Item 65)
The method according to any one of items 46, 62 to 64, wherein the Wnt3a polypeptide is a mammalian Wnt3a polypeptide.
(Item 66)
65. The method of item 65, wherein the mammal is a human.
(Item 67)
46. The method of item 46, wherein the conditioned medium comprises serum or serum substitute.
(Item 68)
67. The method of item 67, wherein the conditioned medium comprises serum.
(Item 69)
68. The method of item 68, wherein the serum is fetal bovine serum.
(Item 70)
69. The method of item 69, wherein the serum concentration is between about 0.1% and about 15% in the conditioned medium.
(Item 71)
67. The method of item 67, wherein the conditioned medium comprises a serum substitute.
(Item 72)
The method of item 71, wherein the serum substitute is a lipid-based substitute.
(Item 73)
46. The method of item 46, wherein the conditioned medium is a serum-free medium.
(Item 74)
The method of any one of items 46-66, wherein the lipid modification is palmitoylation.
(Item 75)
The method of any one of items 46-74, wherein the Wnt3a polypeptide is further glycosylated.
(Item 76)
The method of any one of items 46-75, wherein the Wnt3a product is separated from the sample mixture by centrifugation.
(Item 77)
The method of item 76, wherein the centrifugation time is up to 1 hour at a temperature between about 1 ° C and about 8 ° C.
(Item 78)
The method of any one of items 46-77, wherein the Wnt3a product after centrifugation is resuspended in sterile 1x phosphate buffered saline (PBS).
(Item 79)
The method of any one of items 46-78, wherein the Wnt3a product is stable under nitrogen.
(Item 80)
The method of any one of items 46-79, wherein the Wnt3a product is stable at temperatures between about 1 ° C and about 8 ° C.
(Item 81)
A method for preparing a liposome Wnt polypeptide, which comprises contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, and the phospholipids constituting the liposome have a phase transition temperature of about 10 ° C to about 25 ° C. How to have.
(Item 82)
A method for preparing a liposome Wnt polypeptide, which comprises contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, and the length of the tail carbon of the phospholipid constituting the liposome is about 12 to carbon. A method that is between about 14.
(Item 83)
The method of item 81 or 82, wherein the contacting step is performed at a temperature between about 21 ° C and about 25 ° C.
(Item 84)
The method of item 81 or 82, wherein the contact time is between about 6 hours and about 24 hours.
(Item 85)
The method of item 81 or 82, further comprising a further purification step prior to the step of contacting the Wnt polypeptide with an aqueous solution of liposomes.
(Item 86)
85. The method of item 85, wherein the further purification step is an ion exchange purification step, a hydrophobic purification step, or an affinity purification step.
(Item 87)
86. The method of item 86, wherein the further purification step is an ion exchange purification step.
(Item 88)
87. The method of item 87, wherein the ion exchange purification step comprises contacting the sample with beads on which a sulfonated polycyclic aromatic compound is immobilized or a column on which a sulfonated polycyclic aromatic compound is immobilized.
(Item 89)
87 or 88. The method of item 87 or 88, wherein the ion exchange purification step comprises a step gradient.
(Item 90)
The method according to any one of items 87 to 89, wherein the ion exchange purification buffer comprises a surfactant.
(Item 91)
90. The method of item 90, wherein the surfactant is CHAPS or Triton X-100.
(Item 92)
86. The method of item 86, wherein the further purification step is a hydrophobic purification step.
(Item 93)
92. The method of item 92, wherein the hydrophobic purification step comprises beads with protein A immobilized or a protein A column.
(Item 94)
The method of any one of items 85-93, wherein the yield of the Wnt polypeptide after the further purification step is between about 60% and about 99%.
(Item 95)
The method of any one of items 81-94, further comprising the step of collecting the Wnt polypeptide from a conditioned medium comprising cells derived from an expressing cell line.
(Item 96)
95. The method of item 95, wherein the expression cell line is a mammalian expression cell line.
(Item 97)
The method of item 96, wherein the mammalian expression cell line is a Chinese hamster ovary (CHO) cell line.
(Item 98)
97. The method of item 97, wherein the conditioned medium comprises serum or serum substitute.
(Item 99)
98. The method of item 98, wherein the serum is fetal bovine serum.
(Item 100)
The method of item 99, wherein the serum concentration is between about 0.1% and about 15% in the conditioned medium.
(Item 101)
98. The method of item 98, wherein the conditioned medium comprises a serum substitute.
(Item 102)
101. The method of item 101, wherein the serum substitute is a lipid-based substitute.
(Item 103)
97. The method of item 97, wherein the conditioned medium is a serum-free medium.
(Item 104)
The method according to any one of items 81 to 98, wherein the Wnt polypeptide is a Wnt3a polypeptide.
(Item 105)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 104. The method of item 104, comprising sequence identity of.
(Item 106)
10. The method of item 104, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 107)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The method according to any one of items 104 to 106.
(Item 108)
The method according to any one of items 81 to 107, wherein the Wnt polypeptide is a Wnt5a polypeptide or a Wnt10b polypeptide.
(Item 109)
Items 81-108, wherein the liposome has a net charge of 0 between pH about 6.5 to about 8.0, about 7.0 to about 7.8, or about 7.2 to about 7.6. The method according to any one item.
(Item 110)
The method according to any one of items 81 to 109, wherein the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC).
(Item 111)
The method of any one of items 81-110, wherein the liposome further comprises cholesterol.
(Item 112)
The method of item 110 or 111, wherein the concentration of DMPC and cholesterol is defined by a ratio between about 70:30 and about 100: 0.
(Item 113)
The method according to any one of items 81 to 112, wherein the Wnt polypeptide is a mammalian Wnt polypeptide.
(Item 114)
113. The method of item 113, wherein the mammal is a human.
(Item 115)
The method according to any one of items 81 to 114, wherein the lipid modification is palmitoylation.
(Item 116)
The method of any one of items 81-115, wherein the Wnt polypeptide is further glycosylated.
(Item 117)
The method of any one of items 81-116, wherein the Wnt product is separated from the sample mixture by centrifugation.
(Item 118)
The method of item 117, wherein the centrifugation time is up to 1 hour at a temperature between about 1 ° C and about 8 ° C.
(Item 119)
The method of any one of items 81-118, wherein the Wnt product after centrifugation is resuspended in sterile 1x PBS.
(Item 120)
The method of any one of items 81-119, wherein the Wnt product is stable under nitrogen.
(Item 121)
The method of any one of items 81-120, wherein the Wnt product is stable at temperatures between about 1 ° C and about 8 ° C.
(Item 122)
A composition of isolated enhanced mammalian bone marrow cells, after treatment with liposome Wnt polypeptide, said cells: Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Left1, and A composition having an enhanced expression level of one or more of the biomarkers selected from the group consisting of Tcf4.
(Item 123)
122. The composition of item 122, wherein the mammalian bone marrow cells are rodent bone marrow cells.
(Item 124)
The composition according to item 123, wherein the rodent bone marrow cells are collected from rodents aged 10 months or older.
(Item 125)
122. The composition of item 122, wherein the mammalian bone marrow cells are human bone marrow cells.
(Item 126)
125. The composition of item 125, wherein the human bone marrow cells are obtained from a subject 35 years or older.
(Item 127)
A composition of mammalian bone marrow cells obtained from a human subject 35 years or older, said mammalian bone marrow cells at enhanced expression levels after treatment with liposome Wnt polypeptide, Runx2. , Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4, a composition that expresses one or more of the biomarkers selected from the group.
(Item 128)
The composition according to item 122 or 127, wherein the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4.
(Item 129)
The composition according to item 122 or 127, wherein the biomarker is selected from osteocalcin and osteopontin.
(Item 130)
The composition according to any one of items 122 to 129, wherein the enhanced expression level is compared to untreated mammalian bone marrow cells.
(Item 131)
The composition of item 130, wherein the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells.
(Item 132)
The composition according to any one of items 122 to 131, wherein after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have a reduced expression level of a biomarker selected from SOX9 and PPARγ. ..
(Item 133)
The composition of item 132, wherein the reduced expression level in the mammalian bone marrow cells is compared to untreated mammalian bone marrow cells.
(Item 134)
The composition according to any one of items 122 to 133, wherein said bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells.
(Item 135)
The composition according to item 134, wherein the reduction in the level of apoptosis is about 50%.
(Item 136)
The composition according to any one of items 122 to 135, wherein the mammalian bone marrow cells contain enhanced osteogenic potential as compared to untreated mammalian bone marrow cells.
(Item 137)
136. The composition of item 136, wherein the enhanced bone formation potential comprises a new bone growth level at the site of bone defect after transplantation of treated mammalian bone marrow cells.
(Item 138)
137. The composition of item 137, wherein the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells.
(Item 139)
The new bone growth levels of the transplanted treated mammalian bone marrow cells are about 1% to about 20% or about 5% to about 5% to the new bone growth levels of the transplanted untreated mammalian bone marrow cells. 137 or 138. The composition according to item 137 or 138, which is between 12%.
(Item 140)
The composition according to any one of items 122 to 139, wherein the mammalian bone marrow cells include attached bone marrow cells and non-attached bone marrow cells.
(Item 141)
The composition according to item 140, wherein the mammalian bone marrow cells are adherent bone marrow cells.
(Item 142)
14. The composition of item 141, wherein the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 143)
The composition according to item 141, wherein the attached bone marrow cells include bone marrow stromal cells.
(Item 144)
The composition according to any one of items 122 to 143, wherein the mammalian bone marrow cells are autologous bone marrow cells.
(Item 145)
144. The composition of item 144, wherein the autologous bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 146)
145. The composition of item 145, wherein the autologous bone marrow cells are adherent bone marrow cells.
(Item 147)
146. The composition of item 146, wherein the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 148)
146. The composition of item 146, wherein the autologous bone marrow cells include bone marrow stromal cells.
(Item 149)
The composition according to any one of items 122 to 143, wherein the mammalian bone marrow cells are allogeneic bone marrow cells.
(Item 150)
149. The composition of item 149, wherein the allogeneic bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 151)
The composition according to item 150, wherein the allogeneic bone marrow cells are adherent bone marrow cells.
(Item 152)
151. The composition of item 151, wherein the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 153)
151. The composition of item 151, wherein the allogeneic bone marrow cells include bone marrow stromal cells.
(Item 154)
129. The composition of item 126 or 127, wherein the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a human subject younger than 35 years.
(Item 155)
The composition according to any one of items 122 to 154, wherein the liposome Wnt polypeptide contains a Wnt polypeptide and an aqueous solution of the liposome.
(Item 156)
155. The composition according to item 155, wherein the phospholipid constituting the liposome has a phase transition temperature of about 10 ° C to about 25 ° C.
(Item 157)
The composition according to item 155 or 156, wherein the length of the tail carbon of the phospholipid constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 158)
155. The composition of item 155, wherein the Wnt polypeptide is a Wnt3a polypeptide.
(Item 159)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about the amino acid sequence set forth in SEQ ID NO: 1. 158. The composition of item 158, comprising 95% sequence identity.
(Item 160)
158. The composition of item 158, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 161)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The composition according to any one of items 158 to 160.
(Item 162)
155. The composition according to item 155, wherein the Wnt polypeptide is a Wnt5a polypeptide or a Wnt10b polypeptide.
(Item 163)
A mammalian bone marrow composition made by a process comprising contacting isolated mammalian bone marrow cells ex vivo with a liposome Wnt polypeptide, wherein the contact time is from about 30 minutes to about 4 hours. Between, mammalian bone marrow compositions.
(Item 164)
163. The mammalian bone marrow composition according to item 163, wherein the contact temperature is between about 0 ° C. and about 37 ° C., or between about 20 ° C. and about 25 ° C.
(Item 165)
163 or 164. The mammalian bone marrow composition according to item 163 or 164, wherein the process further comprises a washing step of removing the free liposome Wnt polypeptide after the contacting step.
(Item 166)
The mammalian bone marrow composition according to any one of items 163 to 165, wherein the process further comprises the step of transplanting the liposome Wnt polypeptide into a bone defect site.
(Item 167)
One of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Left1, and Tcf4, as compared to untreated mammalian bone marrow cells. Alternatively, the mammalian bone marrow composition according to any one of items 163 to 166, which has a plurality of enhanced expression levels.
(Item 168)
The mammalian bone marrow composition according to any one of items 163 to 166, wherein the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4.
(Item 169)
The mammalian bone marrow composition according to any one of items 163 to 166, wherein the biomarker is selected from osteocalcin and osteopontin.
(Item 170)
The item according to any one of items 167 to 169, wherein the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. Mammalian bone marrow composition.
(Item 171)
163. The mammalian bone marrow according to any one of items 163 to 170, wherein after treatment with the liposome Wnt polypeptide, the mammalian bone marrow cells further have a reduced expression level of a biomarker selected from SOX9 and PPARγ. Composition.
(Item 172)
171. The mammalian bone marrow composition of item 171 wherein the reduced expression levels in the mammalian bone marrow cells are compared to untreated mammalian bone marrow cells.
(Item 173)
The mammalian bone marrow composition according to any one of items 163 to 172, wherein said bone marrow cells further comprise a reduction in the level of apoptosis as compared to untreated mammalian bone marrow cells.
(Item 174)
173. The mammalian bone marrow composition according to item 173, wherein the reduction in the level of apoptosis is about 50%.
(Item 175)
The mammalian bone marrow composition according to any one of items 163 to 174, wherein the mammalian bone marrow cells contain enhanced bone marrow formation potential as compared to untreated mammalian bone marrow cells.
(Item 176)
175. The mammalian bone marrow composition of item 175, wherein the enhanced bone marrow potential comprises a new bone growth level at the site of bone defect after transplantation of treated mammalian bone marrow cells.
(Item 177)
176. The mammalian bone marrow composition of item 176, wherein the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells.
(Item 178)
The new bone growth levels of the transplanted treated mammalian bone marrow cells are about 1% to about 20% or about 5% to about 5% to the new bone growth levels of the transplanted untreated mammalian bone marrow cells. 176 or 177. The mammalian bone marrow composition according to item 176 or 177, which is between 12%.
(Item 179)
The mammalian bone marrow composition according to any one of items 163 to 178, wherein the mammalian bone marrow cells include adherent bone marrow cells and non-adherent bone marrow cells.
(Item 180)
179. The mammalian bone marrow composition according to item 179, wherein the mammalian bone marrow cells are adherent bone marrow cells.
(Item 181)
The mammalian bone marrow composition according to item 180, wherein the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 182)
The mammalian bone marrow composition according to item 180, wherein the attached bone marrow cells include bone marrow stromal cells.
(Item 183)
The mammalian bone marrow composition according to any one of items 163 to 182, wherein the mammalian bone marrow cells are autologous bone marrow cells.
(Item 184)
183. The mammalian bone marrow composition of item 183, wherein the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest.
(Item 185)
183 or 184. The mammalian bone marrow composition according to item 183 or 184, wherein the autologous bone marrow cells include attached bone marrow cells and non-attached bone marrow cells.
(Item 186)
185. The mammalian bone marrow composition according to item 185, wherein the autologous bone marrow cells are adherent bone marrow cells.
(Item 187)
186. The mammalian bone marrow composition according to item 186, wherein the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 188)
186. The mammalian bone marrow composition according to item 186, wherein the autologous bone marrow cells include bone marrow stromal cells.
(Item 189)
The mammalian bone marrow composition according to any one of items 163 to 182, wherein the isolated mammalian bone marrow cell is an allogeneic bone marrow cell.
(Item 190)
189. The mammalian bone marrow composition according to item 189, wherein the allogeneic bone marrow cells include attached bone marrow cells and non-attached bone marrow cells.
(Item 191)
The mammalian bone marrow composition according to item 190, wherein the allogeneic bone marrow cells are adherent bone marrow cells.
(Item 192)
191. The mammalian bone marrow composition according to item 191 in which the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 193)
191. The mammalian bone marrow composition according to item 191 in which the allogeneic bone marrow cells include bone marrow stromal cells.
(Item 194)
The mammalian bone marrow composition according to any one of items 163 to 193, wherein the mammalian bone marrow cells are rodent bone marrow cells.
(Item 195)
194. The mammalian bone marrow composition according to item 194, wherein the rodent bone marrow cells are collected from rodents aged 10 months or older.
(Item 196)
The mammalian bone marrow composition according to any one of items 163 to 193, wherein the mammalian bone marrow cells are human bone marrow cells.
(Item 197)
196. The mammalian bone marrow composition of item 196, wherein the human bone marrow cells are harvested from a 35-year-old or older subject.
(Item 198)
196 or 197. The mammalian bone marrow composition according to item 196 or 197, wherein the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a subject younger than 35 years.
(Item 199)
The mammalian bone marrow composition according to any one of items 163 to 198, wherein the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome.
(Item 200)
199. The mammalian bone marrow composition according to Item 199, wherein the phospholipid constituting the liposome has a phase transition temperature of about 10 ° C. to about 25 ° C.
(Item 201)
199 or 200. The mammalian bone marrow composition according to item 199 or 200, wherein the length of the tail carbon of the phospholipid constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 202)
199. The mammalian bone marrow composition according to item 199, wherein the Wnt polypeptide is a Wnt3a polypeptide.
(Item 203)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 202. The mammalian bone marrow composition of item 202.
(Item 204)
The mammalian bone marrow composition of item 202, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 205)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The mammalian bone marrow composition according to any one of items 202 to 204.
(Item 206)
199. The mammalian bone marrow composition according to item 199, wherein the Wnt polypeptide is a Wnt5a polypeptide or a Wnt10b polypeptide.
(Item 207)
A method of treating a bone defect in a subject
a) Ex vivo contacting a sample containing mammalian bone marrow cells with a liposome Wnt3a polypeptide;
b) With the step of washing the sample to remove the free liposome Wnt3a polypeptide;
c) A method comprising the step of transplanting a bone graft material treated with the liposome Wnt3a polypeptide into a bone defect site.
(Item 208)
207. The method of item 207, wherein the contact temperature is between about 0 ° C. and about 37 ° C., or about 20 ° C. and about 25 ° C.
(Item 209)
207. The method of item 207, wherein the contact time is between about 30 minutes and about 4 hours.
(Item 210)
Among the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Lef1, and Tcf4 in which the bone marrow cells are enhanced as compared with untreated mammalian bone marrow cells. 207. The method of item 207, which has one or more expression levels of.
(Item 211)
207. The method of item 207, wherein the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4.
(Item 212)
207. The method of item 207, wherein the biomarker is selected from osteocalcin and osteopontin.
(Item 213)
Any one of items 207 or 210-212, wherein the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. The method described in.
(Item 214)
207. The method of any one of items 207 or 210-213, wherein after treatment with the liposome Wnt3a polypeptide, said mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. ..
(Item 215)
214. The method of item 214, wherein the reduced expression level in the mammalian bone marrow cells is compared to untreated mammalian bone marrow cells.
(Item 216)
207. The method of any one of items 207 or 210-215, wherein the bone marrow cells further comprise a reduction in the level of apoptosis compared to untreated mammalian bone marrow cells.
(Item 217)
216. The method of item 216, wherein the reduction in apoptosis levels is about 50%.
(Item 218)
207. The method of any one of items 207 or 210-217, wherein the mammalian bone marrow cells contain enhanced osteogenic potential as compared to untreated mammalian bone marrow cells.
(Item 219)
218. The method of item 218, wherein the enhanced bone formation potential comprises a new bone growth level at the site of bone defect after transplantation of treated mammalian bone marrow cells.
(Item 220)
219. The method of item 219, wherein the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells.
(Item 221)
The new bone growth levels of the transplanted treated mammalian bone marrow cells are about 1% to about 20% or about 5% to about 5% to the new bone growth levels of the transplanted untreated mammalian bone marrow cells. 219 or 220. The method of item 219 or 220, which is between 12%.
(Item 222)
207. The method of any one of items 207-221, wherein the mammalian bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 223)
222. The method of item 222, wherein the mammalian bone marrow cells are adherent bone marrow cells.
(Item 224)
223. The method of item 223, wherein the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 225)
223. The method of item 223, wherein the attached bone marrow cells include bone marrow stromal cells.
(Item 226)
The method according to any one of items 207 to 225, wherein the mammalian bone marrow cells are autologous bone marrow cells.
(Item 227)
226. The method of item 226, wherein the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest.
(Item 228)
226 or 227. The method of item 226 or 227, wherein the autologous bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 229)
228. The method of item 228, wherein the autologous bone marrow cells are adherent bone marrow cells.
(Item 230)
229. The method of item 229, wherein the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 231)
229. The method of item 229, wherein the autologous bone marrow cells include bone marrow stromal cells.
(Item 232)
The method according to any one of items 207 to 225, wherein the isolated mammalian bone marrow cell is an allogeneic bone marrow cell.
(Item 233)
232. The method of item 232, wherein the allogeneic bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 234)
233. The method of item 233, wherein the allogeneic bone marrow cells are adherent bone marrow cells.
(Item 235)
234. The method of item 234, wherein the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 236)
234. The method of item 234, wherein the allogeneic bone marrow cells include bone marrow stromal cells.
(Item 237)
The method according to any one of items 207 to 236, wherein the mammalian bone marrow cell is a rodent bone marrow cell.
(Item 238)
237. The method of item 237, wherein the rodent bone marrow cells are harvested from rodents older than 10 months.
(Item 239)
The method according to any one of items 207 to 236, wherein the mammalian bone marrow cell is a human bone marrow cell.
(Item 240)
239. The method of item 239, wherein the human bone marrow cells are harvested from a subject 35 years or older.
(Item 241)
239 or 240. The method of item 239 or 240, wherein the human bone marrow cells after treatment with the liposome Wnt3a polypeptide exhibit the biomarker expression levels observed in a human subject younger than 35 years.
(Item 242)
The method according to any one of items 207 to 241 wherein the liposome Wnt3a polypeptide comprises a Wnt3a polypeptide and an aqueous solution of the liposome.
(Item 243)
242. The method of item 242, wherein the phospholipids constituting the liposome have a phase transition temperature of about 10 ° C to about 25 ° C.
(Item 244)
242 or 243 of item 242 or 243, wherein the length of the tail carbon of the phospholipid constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 245)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 242. The method of item 242, comprising the sequence identity of.
(Item 246)
242. The method of item 242, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 247)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. 242. The method of any one of 245 or 246.
(Item 248)
A method of treating a bone defect in a subject
a) Ex vivo contacting a sample containing mammalian bone marrow cells with a liposome Wnt polypeptide;
b) With the step of washing the sample to remove the free liposome Wnt polypeptide;
c) A method comprising the step of transplanting a bone graft material treated with the liposome Wnt polypeptide into a bone defect site.
(Item 249)
248. The method of item 248, wherein the contact temperature is between about 0 ° C. and about 37 ° C., or about 20 ° C. and about 25 ° C.
(Item 250)
248. The method of item 248, wherein the contact time is between about 30 minutes and about 4 hours.
(Item 251)
One of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Left1, and Tcf4, as compared to untreated mammalian bone marrow cells. 248. The method of item 248, which also has a plurality of enhanced expression levels.
(Item 252)
248. The method of item 248, wherein the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4.
(Item 253)
248. The method of item 248, wherein the biomarker is selected from osteocalcin and osteopontin.
(Item 254)
Any one of items 248 or 251 to 253, wherein the enhanced expression level is between about 2% to about 20% or about 4% to about 10% as compared to untreated mammalian bone marrow cells. The method described in.
(Item 255)
248 or 251 to 254, wherein after treatment with the liposome Wnt polypeptide, said mammalian bone marrow cells further have reduced expression levels of biomarkers selected from SOX9 and PPARγ. Method.
(Item 256)
255. The method of item 255, wherein the reduced expression level in the mammalian bone marrow cells is compared to untreated mammalian bone marrow cells.
(Item 257)
248. The method of any one of items 248 or 251 to 256, wherein said bone marrow cells further comprise a reduction in the level of apoptosis compared to untreated mammalian bone marrow cells.
(Item 258)
257. The method of item 257, wherein the reduction in apoptosis levels is about 50%.
(Item 259)
248. The method of any one of items 248 or 251 to 258, wherein the mammalian bone marrow cells contain enhanced osteogenic potential as compared to untreated mammalian bone marrow cells.
(Item 260)
259. The method of item 259, wherein the enhanced bone formation potential comprises a new bone growth level at the site of bone defect after transplantation of treated mammalian bone marrow cells.
(Item 261)
260. The method of item 260, wherein the new bone growth level is compared to the new bone growth level of transplanted untreated mammalian bone marrow cells.
(Item 262)
The new bone growth levels of the transplanted treated mammalian bone marrow cells are about 1% to about 20% or about 5% to about 5% to the new bone growth levels of the transplanted untreated mammalian bone marrow cells. 261. The method of item 260 or 261, which is between 12%.
(Item 263)
The method according to any one of items 248 to 262, wherein the mammalian bone marrow cells include attached bone marrow cells and non-attached bone marrow cells.
(Item 264)
263. The method of item 263, wherein the mammalian bone marrow cells are adherent bone marrow cells.
(Item 265)
264. The method of item 264, wherein the attached bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 266)
264. The method of item 264, wherein the attached bone marrow cells include bone marrow stromal cells.
(Item 267)
The method according to any one of items 248 to 266, wherein the mammalian bone marrow cell is an autologous bone marrow cell.
(Item 268)
267. The method of item 267, wherein the autologous bone marrow cells are harvested from the femur, tibia, and / or iliac crest.
(Item 269)
The method according to any one of items 267 to 268, wherein the autologous bone marrow cells include attached bone marrow cells and non-attached bone marrow cells.
(Item 270)
269. The method of item 269, wherein the autologous bone marrow cells are adherent bone marrow cells.
(Item 271)
270. The method of item 270, wherein the autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 272)
270. The method of item 270, wherein the autologous bone marrow cells include bone marrow stromal cells.
(Item 273)
The method according to any one of items 248 to 266, wherein the isolated mammalian bone marrow cell is an allogeneic bone marrow cell.
(Item 274)
273. The method of item 273, wherein the allogeneic bone marrow cells include adherent and non-adherent bone marrow cells.
(Item 275)
274. The method of item 274, wherein the allogeneic bone marrow cells are adherent bone marrow cells.
(Item 276)
275. The method of item 275, wherein the allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells.
(Item 277)
275. The method of item 275, wherein the allogeneic bone marrow cells include bone marrow stromal cells.
(Item 278)
The method according to any one of items 248 to 277, wherein the mammalian bone marrow cell is a rodent bone marrow cell.
(Item 279)
278. The method of item 278, wherein the rodent bone marrow cells are harvested from rodents older than 10 months.
(Item 280)
The method according to any one of items 248 to 277, wherein the mammalian bone marrow cell is a human bone marrow cell.
(Item 281)
280. The method of item 280, wherein the human bone marrow cells are harvested from a subject 35 years or older.
(Item 282)
280 or 281. The method of item 280 or 281 wherein the human bone marrow cells after treatment with a liposome Wnt polypeptide exhibit the biomarker expression levels observed in a subject younger than 35 years.
(Item 283)
The method according to any one of items 248 to 282, wherein the liposome Wnt polypeptide comprises a Wnt polypeptide and an aqueous solution of the liposome.
(Item 284)
283. The method of item 283, wherein the phospholipids constituting the liposome have a phase transition temperature of about 10 ° C to about 25 ° C.
(Item 285)
283 or 284. The method of item 283 or 284, wherein the length of the tail carbon of the phospholipid constituting the liposome is between about 12 carbons and about 14 carbons.
(Item 286)
283. The method of item 283, wherein the Wnt polypeptide is a Wnt3a polypeptide.
(Item 287)
The Wnt3a polypeptide is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the amino acid sequence set forth in SEQ ID NO: 1. 286. The method of item 286, comprising sequence identity of.
(Item 288)
286. The method of item 286, wherein the Wnt3a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 289)
The Wnt3a polypeptide is lipid-modified at the amino acid position corresponding to the amino acid residue 209 shown in SEQ ID NO: 1 and not lipid-modified at the amino acid position corresponding to the amino acid residue 77 shown in SEQ ID NO: 1. The method according to any one of items 286 to 288.
(Item 290)
283. The method of item 283, wherein the Wnt polypeptide is a Wnt5a polypeptide or a Wnt10b polypeptide.
(Item 291)
A kit for making a bone graft material containing the liposome Wnt polypeptide according to items 23 to 45.
(Item 292)
A kit for making a bone graft material containing the liposome Wnt3a polypeptide according to items 1 to 22.
(Item 293)
A kit for making a bone graft material comprising the composition of the isolated enhanced mammalian bone marrow cells according to items 127-162.

Further embodiments and embodiments will also be apparent from the rest of the disclosure and are included in the present invention.

FIG. 1 illustrates the purification scheme of the present invention.

FIG. 2 points to SDS-PAGE analysis and staining analysis with Coomassie blue for Wnt3a.

FIG. 3 illustrates mass spectrometric data.

FIG. 4 illustrates the characterization of L-Wnt3a by TEM. L-Wnt3a was visualized using TEM and the average diameter of vesicles was calculated.

FIG. 5 illustrates the quantification of human Wnt3a activity and L-Wnt3a activity. Bioluminescence was quantified by triple reading on a dual-light ready luminometer (Berthold). The activity of human Wnt3a (ng / uL; A) and L-Wnt3a (B) was defined from the calibration curve prepared by serial dilution of Wnt3a protein.

FIG. 6 shows the stability of L-Wnt3a at 4 ° C. The interaction between liposomes and Wnt3a was investigated. Liposomes Wnt3a (L-Wnt3a) were incubated at 4 ° C. for up to 48 days. The human L-Wnt3a polypeptide was then examined for activity in the LSL assay. The L-Wnt3a polypeptide showed no loss of activity over the 48-day test period.

FIG. 7 shows the stability of L-Wnt3a at 23 ° C.

FIG. 8 illustrates the stability of L-Wnt3a at 37 ° C. The association of human Wnt3a with DMPC: cholesterol liposomes was investigated to determine if it would provide stability to the Wnt3a polypeptide. Applying data from multiple time points to a single exponential decay, after 10 hours, L-Wnt3a retains half of its activity at 37 ° C. (blue line, FIG. 8A), but Wnt3a. Was shown to lose its activity within 5 minutes (red line, FIG. 8A). Loss of activity in human Wnt3a was due to proteolysis. In the Wnt3a + CHAPS solution, a low molecular weight band was detectable by immunoblot (Fig. 8B). Immunoblots of the L-Wnt3a preparation did not detect a low molecular weight band corresponding to Wnt3a (FIG. 8C).

FIG. 9 illustrates human Wnt3a polypeptides produced from cells grown in suspension cultures in comparison with human Wnt3a polypeptides produced from cells grown in adherent cultures.

FIG. 10 illustrates the induction of human Wnt3a polypeptide secretion in a doxycycline concentration-dependent manner. Doxycycline was added to the medium at a concentration (ng / uL) that affected the amount of human Wnt3a produced by CHO cells.

FIG. 11 illustrates the use of fetal bovine serum for the secretion of human Wnt3a polypeptide from CHO cells.

FIG. 12 describes the secretion of human Wnt3a from CHO cells as a function of the number of days in culture.

FIG. 13 illustrates the robustness, accuracy, detection limit, and specificity of the LSL cell reporter assay.

FIG. 14 illustrates a dose-response curve for L-Wnt3a using primary cells. (A) Within mouse embryonic fibroblasts (MEFs), the linear range of effective concentrations was 0.025-0.1 ng / μL human Wnt3a. (B) It is a figure showing the L-Wnt3a activity in bone marrow-derived stem cells, and it was determined from this that the linear range of effective concentration ranges from 0.004 to 0.08 ng / μL.

FIG. 15 illustrates the activity of human Wnt3a under different lipid formulations. When CHAPS was replaced with DMPC and cholesterol lipids, the activity of Wnt3a was maintained, but liposomes made with lipids such as MPPC, DPPC, DMPS, DMPG, and DMGE were inactive.

FIG. 16 illustrates the density gradient of sucrose.

FIG. 17 illustrates the affinity of human Wnt3a for liposome membranes. The binding affinity between Wnt3a / Frizzled was about 3.6 nM. The binding affinity between Wnt3a / LRP6 was about 9 nM. The binding affinity between Wnt3a and the liposome membrane was approximately 6 nM based on a pull-down assay between Wnt3a, Frizzled (Fz), and Lrp6.

FIG. 18 illustrates the reaction rate of human Wnt3a associating with liposomes.

FIG. 19 illustrates the binding of Wnt5a and Wnt10b polypeptides to neutral DMPC liposomes.

FIG. 20 illustrates the individual components of the bone graft material. Bone graft material contains a stem cell population and a progenitor cell population. FIG. 20A points to Gomori staining for BGM taken from the rat femur, (FIG. 20B) points to Gomori staining for BGM taken from the rat iliac crest, and FIG. 20C points to Gomori staining for BGM taken from the rat iliac crest (FIG. 20C). Indicates the iliac stain on the collected BGM. FIG. 20D shows a quantitative RT-PCR analysis of the expression of endogenous bone-forming genes in rat BGM freshly harvested from the indicated sources. FIG. 20E outlines an experimental design for transplanting autologous BGM into rat SRC. FIG. 20F illustrates a representative tissue section of iliac crest BGM 7 days after transplantation, stained to detect BrdU uptake. The dotted line points to a small piece of trabecular bone contained within the BGM. 20G, 20H, and 20I are expressions of Runx2, Sox9, and PPARγ, respectively. FIG. 20J shows a representative tissue section of BGM stained with aniline blue to detect the osteoid matrix, and Asterisk contrasts the new bone matrix with the old bone fragments (yellow dotted line). Point to. In this panel and in Figure 20G, the renal surface is indicated by a white dotted line. FIG. 20K is a histology by safranin O / Fast green that detects proteoglycan-rich cartilage (red), and FIG. 20L is a gomoritrichrome stain that detects adipocytes. Abbreviation: BrdU: Bromodeoxyuridine; PPARγ: Peroxisome proliferator-activated protein gamma. Scale bar: 50 μm, asterisk: p <0.05.

FIG. 21 describes the bone graft material as Wnt responsive. FIG. 21A points to GFP ^{+ ve cells in Axin2 CreERT2} ; R26 ^mTmG mice visualized by immunostaining of the periosteum, FIG. 21B is the endosteum. ^{FIG. 21C is a quantification of GFP + ve} cells / whole cells within a designated microscopic field of view. FIG. 21D indicates that GFP ^{+ ve} cells in BGM were visualized by fluorescence. FIG. 21E shows quantitative absolute RT-PCR results for endogenous Axin2 expression, endogenous Lef1 expression, and endogenous GAPDH expression ^{in BGM young} (green bar) and BGM ^{aged (gray bar).} FIG. 21F illustrates Western blot analysis for Wnt3a, total beta-catenin, Axin2, and beta-actin ^{in BGM young} (green bar) and BGM ^{aged (gray bar).} Scale bar = 50 μm. Asterisk: p <0.05.

FIG. 22 shows that the bone differentiation potential of BGM decreases with age. FIG. 22A points to a quantitative RT-PCR analysis of the expression of alkaline phosphatase, Osterix, and osteocalcin ^{in BGM young} (green bar) and BGM ^{aged (gray bar).} FIG. 22B is a BGM taken from ACTB-eGFP mice, transplanted into an SRC and visualized in a bright field, and FIG. 22C is the detection of GFP signals in the BGM utilizing fluorescence. FIG. 22D is a representative tissue section (N = 5) stained with aniline blue (insertion view) derived from ^{BGM young} , and (FIG. 22E) is stained with aniline blue derived from ^{BGM aged (FIG. 22E).} (Insert view) is a representative tissue section (N = 5). The dotted line points to the surface of the kidney. FIG. 22F points to a tissue morphometry analysis ^{of aniline blue + v} pixels in the entire area occupied by BGM on day 7 after transplantation. 22G and 22H indicate representative tissue sections stained to detect ALP activity from (G) BGM ^young (N = 5) and (H) BGM ^{aged (N = 5).} FIG. 22I points to the quantification ^{of ALP + ve} pixels in the entire area occupied by BGM on the 7th day after transplantation. 22J and 22K show representative tissue sections immunostained for GFP from (FIG. 22J) BGM ^young (N = 5) and (FIG. 22K) BGM ^{aged (N = 5).} FIG. 22L shows the quantification ^{of GFP + ve} pixels in the entire region occupied by BGM on the 7th day after transplantation. Abbreviation: ALP: Alkaline phosphatase; Occ: Osteocalcin. Scale bar: 100 μm. Asterisk: p <0.05 and double asterisk: p <0.01.

FIG. 23 shows that bone differentiation of BGM requires an endogenous Wnt signal. FIG. 23A shows representative tissue sections stained for ALP activity in BGM treated with adenovirus (Ad-Fc) expressing a mouse IgG2α Fc fragment, or (FIG. 23B) is a soluble Wnt antagonist. Shown are representative tissue sections stained for ALP activity in BGM treated with adenovirus (Ad-Dkk1) expressing Dkk1. FIG. 23C shows representative tissue sections immunostained for PPARγ in Ad-Fc treated BGM, or (FIG. 23D) is immunostained for PPARγ in Ad-Dkk1 treated BGM. The representative tissue section obtained is shown. FIG. 23E illustrates a representative tissue section immunostained for Dlk1 in Ad-Fc treated BGM, or (FIG. 23F) is immunohistochemical for Dlk1 in Ad-Dkk1 treated BGM. Representative tissue sections stained will be described. FIG. 23G illustrates the reconstruction of micro-CT, which detects bone formation in the BGM-treated defect site treated with Ad-Fc, or (FIG. 23H) was treated with Ad-Dkk1. The reconstruction of micro-CT, which detects bone formation in the BGM-treated defect site, will be described. The original defect is indicated by a red dotted circle. FIG. 23I shows the new bone volume (N = 5) ± SEM calculated from the micro-CT data. FIG. 23J points to aniline blue staining of representative tissue sections derived from Ad-Fc treated BGM-treated defect sites, or (FIG. 23K) shows Ad-Dkk1 treated BGM. Indicates aniline blue staining for representative tissue sections derived from the defect site. FIG. 23L is a diagram pointing to the quantification of new bone volume using tissue morphometry analysis. FIG. 23I shows PPAR-γ expression in Ad-Fc treated BM grafts, or (FIG. 23J) shows PPAR-γ expression in Ad-Dkk1 treated BM grafts. Single asterisk: p <0.05. Scale bars: A to B: 200 μm, C to F, J to K: 50 μm, G to H: 2 mm.

FIG. 24 illustrates that human Wnt3a ^{activates BGM aged} and restores its bone differentiation potential. FIG. 24A is a BGM derived from aged ACTB-eGFP mice, treated with L-PBS or L-WNT3A (0.15 μg / ml) for 1 hour and then targeted by qRT-PCR 24 hours later. BGM assayed for gene expression or rapidly transplanted into SRC for 7 days is indicated. FIG. 24B illustrates multiple changes in Axin2 and Left1 expression during ^{BGM aged} treated with L-PBS (gray bar) or L-WNT3A (blue bar). FIG. 24C shows Western blot analysis for total beta-catenin, Axin2, and beta-actin in ^{BGM aged} treated with L-PBS (gray bar) or L-WNT3A (blue bar). BGM ^aged is derived from samples treated with (FIG. 24D) L-PBS (N = 5) and (FIG. 24E) L-WNT3A (N = 5) after harvesting from SRC on day 4 post-transplantation. Representative tissue sections were stained for BrdU uptake (N = 5). FIG. 24F is a diagram showing the quantification ^{of BrdU + ve} pixels in the microscopic field of view centered on the inside of the bone implant. FIG. 24G illustrates a representative tissue section from a sample treated with L-PBS, stained for BrdU uptake 7 days after transplantation (N = 5), (FIG. 24H) is a representative tissue section derived from a sample treated with L-WNT3A (N = 5) and describes a tissue section stained for BrdU uptake 7 days after transplantation. FIG. 24I is a diagram illustrating the quantification of ^{BrdU + ve pixels in the same manner as described above.} FIG. 24J is a representative tissue section derived from a sample treated with L-PBS, pointing to a tissue section immunostained for Dlk1 expression 7 days after transplantation (N = 5), (FIG. 24K) is a representative tissue section derived from a sample treated with L-WNT3A (N = 5) and points to an immunostained tissue section for Dlk1 expression on day 7 post-transplantation. FIG. 24L is a diagram showing the quantification ^{of Dlk1 + ve} pixels in the entire region occupied by BGM on the 7th day after transplantation. FIG. 24M is a representative tissue section from a sample treated with L-PBS, showing an immunostained tissue section for Occ expression on day 7 post-implantation (N = 5), and ( FIG. 24N) is a representative tissue section derived from a sample treated with L-WNT3A (N = 5), showing an immunostained tissue section for the expression of Occ on the 7th day after transplantation. FIG. 24O is a diagram pointing to the quantification of Occ ^{+ ve} pixels, similar to that described for Dlk1. FIG. 24P shows representative tissue sections stained with aniline blue to detect the osteoid matrix in samples treated with L-PBS (N = 5), (FIG. 24Q) shows L-. Representative tissue sections stained with aniline blue to detect the osteoid matrix in samples treated with WNT3A (N = 5) are shown. FIG. 24R is a diagram showing the quantification of a new bone matrix by tissue morphometry. The abbreviations are as described elsewhere herein. Scale bar: 100 μm. Asterisk: p <0.05; Double asterisk: p <0.01.

FIG. 25 illustrates that L-Wnt3a stimulates BGM stem cells to improve spinal fixation. FIG. 25A shows that human MSC cultures were treated with L-PBS or L-WNT3A at 37 ° C. for the indicated period and the Wnt response was determined using qRT-PCR for Axin2 expression. FIG. 25B shows that mouse SSCs were treated with L-PBS or L-WNT3A at 37 ° C. for 12 hours and the Wnt response was assayed by qRT-PCR for Axin2 expression. FIG. 25C shows quantitative absolute RT-PCR for Axin2 and Ref1 expression in response to incubation over 1 hour at room temperature with L-PBS (dashed line) or L-WNT3A (0.15 μg / mL; blue line). The analysis will be described. Data are expressed as RNA copy number / total RNA content ratio over 24 hours. FIG. 25D shows rat spinous processes exposed through a minimal incision and a standardized volume of autologous BGM from the iliac crest treated with L-PBS or L-WNT3A for 1 hour, then (FIG. 25D). 25E) Indicates that the implant was made between the L4 spinal cord transverse process and the L5 spinal cord transverse process. 25F and 25G indicate to POD2 that micro-CT collection was performed on the grafts (pink) treated with (F) L-PBS and (G) L-WNT3A. FIG. 25H re-collects micro-CT on POD49 to assess bone growth in grafts treated with (FIG. 25H) L-PBS (gray) and (FIG. 25I) L-WNT3A (blue). Point to what you have done. FIG. 25J graphed graft growth for each of the treatment groups as a volume multiple of each graft size in POD2 compared to its size in POD49 (pointed to by the color stated above). Point to. Abbreviation: L4: 4th lumbar vertebra, L5: 5th lumbar vertebra, AP: tip process, SP: spinous process, TP: transverse process, POD: post-surgery to day.

FIG. 26 illustrates the quantification of BGM volume. BGM from the tibia and femur was divided into 5 aliquots of approximately 20 μL each and then DNA concentrations were assessed as a means of quantifying cell density in a heterologous mixture of cells, interstitium, and bone fragments. The DNA concentration in sample # 1 was set to 100, and the relative DNA concentrations of the other four aliquots were determined.

FIG. 27 illustrates the engraftment efficiency of BGM. Treatment with L-Wnt3a improves the engraftment efficiency of BGM. ^{FIG. 27A illustrates GFP immunostaining for BGM aged} after 4 days in SRC and (FIG. 27B) illustrates GFP immunostaining for ^{BGM ACT.} FIG. 27C shows the quantification of ^{GFP + ve} pixels divided by all the pixels occupied by BGM on the 4th day after transplantation. ^{FIG. 27D shows the TUNEL stain for BGM aged} after 4 days in the SRC, and FIG. 27E shows the TUNEL stain for BGM ^ACT . FIG. 27F shows the quantification of ^{TUNEL + ve} cells divided by ^{the total DAPI + ve} pixels in the entire region occupied by BGM on the 4th day after transplantation. ^{FIG. 27G shows the TUNEL stain for BGM aged} after 7 days in the SRC, and FIG. 27H shows the TUNEL stain for BGM ^ACT . FIG. 27I is a diagram showing the quantification of ^{TUNEL + ve} cells divided by the ^{total DAPI + ve} pixels in the total region occupied by BGM on the 7th day after transplantation. FIG. 27J illustrates tissue sections stained for tartrate-resistant acid phosphatase (TRAP) activity to detect osteoclasts in ^{BGM aged} after 7 days in SRC, and ^{FIG. 27K (FIG. 27K) in BGM ACT} . Tartrate-resistant acid phosphatase (TRAP) activity-stained tissue sections that detect osteoclasts are described. FIG. 27L shows the quantification of ^{TRAP + v} pixels in the entire area occupied by BGM on the 7th day after transplantation. The scale bar = 100 μm, where ^** indicates p <0.01.

Detailed Description of the Invention Compositions, methods, processes, and kits for use in making liposome Wnt polypeptides are disclosed herein. Also disclosed herein are compositions, methods, processes, and kits for making bone graft materials. In some embodiments, the composition comprises a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, the phospholipids constituting the liposomes having a phase transition temperature of about 10 ° C to about 25 ° C. Has. In some embodiments, the composition comprises a functionally active mammalian Wnt polypeptide and an aqueous solution containing liposomes, the tail carbon length of the phospholipids constituting the liposomes being from about 12 carbons. Between about 14 carbons. In some embodiments, the composition also comprises a functionally active Wnt3a polypeptide and an aqueous solution containing liposomes, wherein the functionally active Wnt3a polypeptide is amino acid residue 209 set forth in SEQ ID NO: 1. Includes lipid modification at the amino acid position corresponding to.

In some embodiments herein, the method of preparing a liposome Wnt polypeptide comprises contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, and the amount of phospholipids constituting the liposome is about 10. Methods having a phase transition temperature of ° C. to about 25 ° C. are described. In some embodiments herein, a method of preparing a liposome Wnt polypeptide, comprising contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, comprising the step of contacting the tail carbon of the phospholipids constituting the liposome. Methods are described in which the length is between about 12 carbons and about 14 carbons. In some embodiments herein, there is also a method of preparing a liposome Wnt3a polypeptide, in which (a) the Wnt3a polypeptide is harvested from a conditioned medium containing Chinese hamster ovary (CHO) cells; b) The step of introducing the Wnt3a polypeptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized; and (c) the step of eluting the Wnt3a polypeptide from the ion exchange column using a step gradient; (D) A method comprising contacting the Wnt3a polypeptide with an aqueous solution of the liposome according to step (c) is also described.

In some embodiments herein, it is a composition of isolated enhanced mammalian bone marrow cells, which, after treatment with liposome Wnt polypeptide, by cells, Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase. , A composition in which the expression level of one or more of the biomarkers selected from the group consisting of type I collagen, Axin2, Leaf1, and Tcf4 is enhanced. In some embodiments herein, it is also a composition of mammalian bone marrow cells obtained from a human subject 35 years or older, after treatment with the liposome Wnt polypeptide. A composition in which cells express one or more of biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. Things are also listed.

In some embodiments herein, the process comprises contacting the isolated mammalian bone marrow cells ex vivo with the liposome Wnt polypeptide, the contact time being from about 30 minutes to about 4 hours. Mammalian bone marrow compositions made by the intervening process are described.

In some embodiments herein, a method of treating a bone defect in a subject, the step of (a) contacting a sample containing mammalian bone marrow cells ex vivo with a liposome Wnt polypeptide; A method comprising (b) washing the sample to remove the free liposome Wnt polypeptide and (c) implanting the bone marrow graft material treated with the liposome Wnt polypeptide into the bone defect site is described. NS. Also herein is a method of treating a bone defect in a subject, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome Wnt3a polypeptide; (b) the sample. Also described is a method comprising washing to remove the free liposome Wnt3a polypeptide; (c) implanting the bone graft material treated with the liposome Wnt3a polypeptide into the bone defect site.

In some embodiments herein, a method of making bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome Wnt polypeptide; A method comprising (b) washing the sample to remove the free liposome Wnt polypeptide and (c) implanting the bone graft material treated with the liposome Wnt polypeptide into the bone defect site is described. .. Also herein is a method of making bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome Wnt3a polypeptide; (b) the sample is washed. Also described is a method comprising removing the free liposome Wnt3a polypeptide; (c) implanting the bone graft material treated with the liposome Wnt3a polypeptide into the bone defect site.

In some aspects of the specification, a non-human mammal of SEQ ID NO: 1, a functionally active Wnt3a polypeptide that is lipid-modified in Ser209 or in the absence of lipid modification in Cys77. Lipid-modified in the corresponding, conserved Ser of the Wnt3a polypeptide; or at least about of the lipid-modified Wnt3a polypeptide in the corresponding, conserved Cys of the non-human mammalian Wnt3a polypeptide. Compositions comprising 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% are described. In some embodiments herein, Wnt3A is a functionally active human Wnt polypeptide other than Wnt3A in the absence of lipid modification to Wnt3A Cys77 in the corresponding conserved Cys. At least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90% of the lipid-modified human Wnt polypeptides in the conserved Ser corresponding to Ser209. , Or a composition comprising at least about 95%.

In some embodiments, the Wnt polypeptide is Wnt3a or a functionally active variant thereof. In another aspect, the Wnt polypeptide is human Wnt3a or a functionally active variant thereof. In yet another aspect, the Wnt polypeptide is Wnt3a of SEQ ID NO: 1 or a functionally active variant thereof. In a further aspect, the lipid modification is palmitoylation. In another aspect, the Wnt polypeptide is a Wnt1 polypeptide, a Wnt2 polypeptide, a Wnt2b (or Wnt13) polypeptide, a Wnt3 polypeptide, a Wnt4 polypeptide, a Wnt5a polypeptide, a Wnt5b polypeptide, a Wnt6 polypeptide, a Wnt7a polypeptide, Wnt7b polypeptide, Wnt8a polypeptide, Wnt8b polypeptide, Wnt9a (Wnt14 or Wnt14b) polypeptide, Wnt9b (Wnt14b or Wnt15) polypeptide, Wnt10a polypeptide, Wnt10b (or Wnt12) polypeptide, Wnt11 polypeptide, Wnt- Human Wnt proteins other than Wnt3A, such as 16a polypeptides, Wnt-16b polypeptides or functionally active variants thereof. In some embodiments, the polypeptide is Wnt5A or Wnt10b or a functionally active variant thereof. In some such embodiments, the Wnt polypeptide is formulated into liposomes, such as liposomes containing DMPC and / or cholesterol.

In another aspect herein, the Wnt polypeptide is, without limitation, a method for purifying from a mammalian cell culture comprising recombinant Chinese hamster ovary (CHO) cells, eg, gel. In the absence of an alternative purification step, such as a filtration purification step, a separation step, such as a separation step on a chromatography column (eg, a Blue Sepharose ion exchange column), is utilized to utilize the Wnt polypeptide-containing cell. A method comprising the step of subjecting the culture medium to purification is described. In some embodiments, purification is also performed in the absence of a purification step with a heparin sulfate column. In another embodiment, the cells are adherent cells and the culture medium further comprises serum such as fetal bovine serum (FBS). In a further embodiment, purification on a column such as a Blue Sepharose ion exchange column is performed using a salt gradient of 150 mM to 1.5 M and the salt can be, for example, sodium chloride or potassium chloride. do. Optionally, the purification scheme is followed by, for example, a purification step using liposomes that are prefabricated and can contain lipids such as DPMC and / or cholesterol, eg, in a molar: molar ratio of 90:10. , Perform further purification steps. In some cases, the use of prefabricated liposomes takes advantage of the hydrophobicity of the Wnt polypeptide to eliminate the need for further purification steps, such as gel filtration purification steps and / or purification steps with a heparin sulfate column.

If the cells are suspended cells, the culture can be performed under serum-free conditions. In some embodiments, the Wnt polypeptide may be a human Wnt3a, eg, Wnt3a such as human Wnt3a of SEQ ID NO: 1 or a functionally active variant thereof, and a human Wnt polypeptide other than Wnt3A, eg, Wnt1 polypeptide, Wnt2 polypeptide, Wnt2b (or Wnt13) polypeptide, Wnt3 polypeptide, Wnt4 polypeptide, Wnt5a polypeptide, Wnt5b polypeptide, Wnt6 polypeptide, Wnt7a polypeptide, Wnt7b polypeptide, Wnt8a polypeptide, Wnt8b polypeptide Peptide, Wnt9a (Wnt14 or Wnt14b) polypeptide, Wnt9b (Wnt14b or Wnt15) polypeptide, Wnt10a polypeptide, Wnt10b (or Wnt12) polypeptide, Wnt11 polypeptide, Wnt-16a polypeptide, Wnt-16b polypeptide, Alternatively, they may be functionally active variants. In some embodiments, the polypeptide is Wnt5A or Wnt10b or a functionally active variant thereof. In a further aspect, the invention relates to a Wnt composition purified by the method described above. In one embodiment, the Wnt composition comprises a Wnt polypeptide that is Wnt3a or a functionally active variant thereof. In another embodiment, the Wnt composition comprises a Wnt polypeptide that is a human Wnt3a or a functionally active variant thereof. In yet another embodiment, the Wnt composition comprises a Wnt polypeptide of SEQ ID NO: 1 human Wnt3a or a functionally active variant thereof.

Some aspects of the specification are also kits for making bone graft materials, the composition of liposome Wnt polypeptide, liposome Wnt3a polypeptide, or isolated enhanced mammalian bone marrow cell. Included kits are also included.

A. Definitions Prior to describing this method, it should be understood that the invention is not limited to the particular method described, as such methods are of course subject to change. Further, since the scope of the present invention is limited only to the scope of the accompanying claims, the terminology used in the present specification is intended to describe only a specific embodiment. It should also be understood that it is not intended to be limited.

If a range of values is presented, each intermediate value between the upper and lower bounds of the range, up to one tenth of the unit of the lower bound, unless the context explicitly indicates otherwise. It is understood that and any other declared value or intermediate value within its stated range is subsumed in the present invention. The upper and lower limits of these subranges are independently contained within the subrange included in the present invention and are determined by any specifically excluded limits within the stated range. The ranges and quantities used herein can also be expressed as "about" specific values or ranges. About also includes just the amount. Therefore, "about 5 μL" means "about 5 μL", but also means "5 μL". In general, the term "about" includes an amount that is expected to be within ± 10% of the amount.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, J. Wiley & Sons (New York, NY, 1994) provide those skilled in the art with general guidance for many of the terms used in this application.

Wnt polypeptides are highly conserved secretory signaling molecules that form a family of signaling molecules that regulate cell-cell interactions during embryogenesis. The terms "Wnt" or "Wnt gene product" or "Wnt polypeptide" as used herein refer to a naturally occurring Wnt polypeptide, Wnt polypeptide fragment, chimeric Wnt polypeptide, or function as described above. Inclusion of actively active mutants. As used herein, the terms "WNT" and "Wnt" are used interchangeably. Similarly, the terms "WNT3A", "WNT3a", "Wnt3A", or "Wnt3a" are also used interchangeably herein.

A "natural sequence" polypeptide is a polypeptide having the same amino acid sequence as a naturally occurring Wnt polypeptide. Polypeptides of such a naturally occurring sequence can be isolated from cells producing endogenous Wnt proteins and can also be made by recombinant or synthetic means. Thus, naturally occurring sequences of polypeptides may be, for example, naturally occurring human polypeptides, mouse polypeptides, or any other mammalian or non-mammalian species, such as the genus Drosophila, C.I. It may have an amino acid sequence of a polypeptide derived from elegans or the like. Optionally, the "natural sequence" polypeptide also comprises an N-terminal methionine. In some cases, "natural sequence" polypeptides do not contain N-terminal methionine.

The term "naturally occurring Wnt polypeptide" includes, without limitation, a mammalian Wnt polypeptide such as a human Wnt polypeptide or a mouse Wnt polypeptide. Human Wnt polypeptides include Wnt1 polypeptide, Wnt2 polypeptide, Wnt2b (or Wnt13) polypeptide, Wnt3 polypeptide, Wnt3a polypeptide, Wnt4 polypeptide, Wnt5a polypeptide, Wnt5b polypeptide, Wnt6 polypeptide, Wnt7a polypeptide, Wnt7b polypeptide, Wnt8a polypeptide, Wnt8b polypeptide, Wnt9a (Wnt14 or Wnt14b) polypeptide, Wnt9b (Wnt14b or Wnt15) polypeptide, Wnt10a polypeptide, Wnt10b (or Wnt12) polypeptide, Wnt11 polypeptide, Wnt- Includes 16a polypeptide, or Wnt-16b polypeptide. Wnt1 can be referred to by Genbank reference number NP0054211.1 and AAH7479.1. Wnt2 can be referred to by Genbank reference number NP003382.1. In general, Wnt2 can be expressed in the thalamus of the brain, in the fetal and adult lungs, and in the placenta. Wnt2B has two isoforms whose Genbank reference numbers are NP004176.2 and NP078613.1, respectively. Isoform 1 can be expressed in the adult heart, brain, placenta, lungs, prostate, testis, ovary, small intestine, and / or colon. In the adult brain, isoform 1 is found primarily in the caudate nucleus, subthalamic nucleus, and thalamus. In some cases, Isoform 1 is also detected in the fetal brain, lungs, and kidneys. Isoform 2 can be expressed in the fetal brain, in the fetal lung, in the fetal kidney, in the caudate nucleus, in the testis, and / or in the cancer cell line.

Wnt3 and Wnt3a play significantly different roles in intercellular signal transduction during neural tube morphogenesis during development. Wnt3 has Genbank reference number AB060284.1 (see also GenBank number BAB61052.1. And AAI03924.1). Wnt3a has the amino acid sequence shown in SEQ ID NO: 1 and the nucleic acid sequence shown in SEQ ID NO: 2. The amino acid and nucleotide sequences of native human Wnt3a are also disclosed at Genbank reference number NM_033131, and the protein sequence is disclosed at Genbank reference number NP_149122. The precursor of the Wnt3a protein has Genbank reference number NP_149122.1. Natural Wnt3a is the length of 352 amino acid residues. Natural Wnt3a contains a signal peptide derived from amino acid residues 1-18. The mature protein contains amino acid residues 19-352. For example, references to Cys77 or Ser209 of Wnt3A as used herein are made in the light of the full-length sequence set forth in SEQ ID NO: 1. In some cases, the term "natural human Wnt3a" polypeptide refers to a polypeptide of SEQ ID NO: 1, a natural human Wnt3a polypeptide with or without its N-terminal methionine (Met), and a natural signal sequence. Refers to a polypeptide with or without it.

Wnt4 has Genbank reference number NP1103880.2 and BAC23080.1. Wnt5a has Genbank reference numbers NP003383.1 and NP003383.2. Wnt5b has Genbank reference numbers BAB62039.1 and AAG38659. Wnt6 has Genbank reference number NP006513.1 and BAB55603.1. Wnt7a has Genbank reference number NP004616.2 and BAA8259.1. Wnt7a can be expressed in the placenta, in the kidney, in the testis, in the uterus, in the fetal lung, in the fetal brain or in the adult brain. Wnt7b has Genbank reference number NP478679.1 and BAB683991. Wnt7b can be expressed in the fetal brain, lungs, and / or kidneys, or in the adult brain, lungs, and / or prostate. Wnt8A has at least two alternative transcripts, Genbank reference number NP114139.1 and NP490645.1. Wnt8B can be expressed in the forebrain. Wnt8B has Genbank reference number NP003384.1. Wnt10A has Genbank reference number AAG45153 and NP0794922.2. Wnt10B is detected in most adult tissues and has the highest levels in the heart and skeletal muscle. Wnt10B has Genbank reference number NP003385.2. Wnt11 can be expressed in the fetal lung, kidney, adult heart, liver, skeletal muscle, and pancreas. Wnt11 has Genbank reference number NP004617.2. Wnt14 has Genbank reference number NP003386.1. Wnt15 can be expressed in the fetal kidney or in the adult kidney. Wnt15 can also be expressed in the brain. Wnt15 has Genbank reference number NP003387.1. Wnt16 has two isoforms, Wnt-16a and Wnt-16b, and is produced by alternative splicing. The isoform Wnt-16a can be expressed in the pancreas. The isoform Wnt-16b can be expressed in peripheral lymphatic organs such as the spleen, appendix, and lymph nodes, or in the kidney. However, Wnt-16b cannot be expressed in the bone marrow. Genbank reference numbers are NP476509.1 and NP057171.2 for Wnt16a and Wnt16b, respectively. All GenBank sequences, SwissProt sequences, and sequences from other databases listed are explicitly incorporated herein by reference.

A "variant" polypeptide means a functionally active polypeptide, defined below, that has less than 100% sequence identity with a naturally occurring polypeptide. .. Such variants are, for example, longer or longer, with one or more amino acid residues added to the N-terminus or C-terminus of the natural sequence or inside, or about 1-300 amino acid residues deleted. Contains shorter polypeptides. A variant polypeptide is a polypeptide with one or more amino acid substitutions, or a derivative of a polypeptide sequence, as compared to a naturally occurring polypeptide sequence, and the resulting product is a non-naturally occurring amino acid. Includes derivatives in which amino acid residues are covalently modified to have.

A "functionally active" Wnt polypeptide (eg, a Wnt3a polypeptide) retains effector function that is directly or indirectly triggered or fulfilled by a naturally occurring Wnt polypeptide. The effector function of the naturally occurring Wnt polypeptide includes stabilization of β-catenin, stimulation of stem cell self-renewal, transformation of C57MG in the Xenopus animal cap assay and induction of target genes, as well as targeting in human teratoma cells. Includes gene expression. Purified Wnt compositions are used in a variety of therapeutic methods, including stem cell maintenance and proliferation, tissue regeneration, and the like.

In some cases, functionally active Wnt variants are at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about about of the native sequence Wnt polypeptide. It will have an amino acid sequence with 80%, at least about 90%, at least about 95%, or at least about 99% amino acid sequence identity. In one embodiment, the native sequence Wnt polypeptide is a mammalian (eg, human) Wnt polypeptide, such as a mammalian (eg, human) Wnt3a.

The term "amino acid" refers to a molecule that contains both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D-isomer and the L-isomer of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, without limitation, α-amino acids, natural amino acids, unnatural amino acids, and amino acid analogs.

The term "α-amino acid" refers to a molecule that contains both an amino group and a carboxyl group and is bound to a carbon called α-carbon.

The term "β-amino acid" refers to a molecule that contains both an amino group and a carboxyl group and is in a β configuration.

The term "naturally occurring amino acids" is commonly found in naturally synthesized peptides and is a single letter abbreviation for A, R, N, C, D, Q, E, G, H, I, L. , K, M, F, P, S, T, W, Y, and V refers to any one of the 20 amino acids known.

The table below outlines the properties of natural amino acids.

"Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acids" are glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. A "charged amino acid" is lysine, arginine, histidine, aspartic acid, glutamic acid, and analogs thereof.

The term "amino acid analog" refers to a molecule that is structurally similar to an amino acid and can substitute for an amino acid when forming a macrocycle of a peptide mimetic. Amino acid analogs are, without limitation, β-amino acids and amino or carboxy groups substituted with similar reactive groups (eg, primary amines substituted with secondary or tertiary amines, or carboxy. Includes amino acids (substitution by ester) of the group.

The term "unnatural amino acid" is commonly found in naturally synthesized peptides and is a single letter abbreviation for A, R, N, C, D, Q, E, G, H, I, L, Refers to an amino acid that is not one of the 20 amino acids known as K, M, F, P, S, T, W, Y, and V. Unnatural amino acids or amino acid analogs are, without limitation,

に従う構造を含む。

Includes structures that follow.

Amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs are cyclic β-amino acid analogs; β-alanine; (R) -β-phenylalanine; (R) -1,2,3,4-tetrahydro-isoquinolin-3-acetic acid; (R). ) -3-Amino-4- (1-naphthyl) -butyric acid; (R) -3-amino-4- (2,4-dichlorophenyl) butyric acid; (R) -3-amino-4- (2-chlorophenyl) -Birate; (R) -3-amino-4- (2-cyanophenyl) -butyric acid; (R) -3-amino-4- (2-fluorophenyl) -butyric acid; (R) -3-amino-4 -(2-Frill) -butyric acid; (R) -3-amino-4- (2-methylphenyl) -butyric acid; (R) -3-amino-4- (2-naphthyl) -butyric acid; (R)- 3-Amino-4- (2-thienyl) -butyric acid; (R) -3-amino-4- (2-trifluoromethylphenyl) -butyric acid; (R) -3-amino-4- (3,4-) Dichlorophenyl) butyric acid; (R) -3-amino-4- (3,4-difluorophenyl) butyric acid; (R) -3-amino-4- (3-benzothienyl) -butyric acid; (R) -3-amino -4- (3-Chlorophenyl) -butyric acid; (R) -3-amino-4- (3-cyanophenyl) -butyric acid; (R) -3-amino-4- (3-fluorophenyl) -butyric acid; R) -3-amino-4- (3-methylphenyl) -butyric acid; (R) -3-amino-4- (3-pyridyl) -butyric acid; (R) -3-amino-4- (3-thienyl) ) -Birate; (R) -3-amino-4- (3-trifluoromethylphenyl) -butyric acid; (R) -3-amino-4- (4-bromophenyl) -butyric acid; (R) -3- Amino-4- (4-chlorophenyl) -butyric acid; (R) -3-amino-4- (4-cyanophenyl) -butyric acid; (R) -3-amino-4- (4-fluorophenyl) -butyric acid; (R) -3-amino-4- (4-iodophenyl) -butyric acid; (R) -3-amino-4- (4-methylphenyl) -butyric acid; (R) -3-amino-4- (4) -Nitrophenyl) -butyric acid; (R) -3-amino-4- (4-pyridyl) -butyric acid; (R) -3-amino-4- (4-trifluoromethylphenyl) -butyric acid; (R)- 3-Amino-4-pentafluoro-phenylbutyric acid; (R) -3-amino-5-hexenoic acid; (R) -3-amino-5-hexic acid; (R) -3-amino-5-phenylpentane Acid; (R) -3-amino-6-phenyl-5-hexenoic acid; (S) -1,2,3,4-tetrahydro-isoquino Phosphorus-3-acetic acid; (S) -3-amino-4- (1-naphthyl) -butyric acid; (S) -3-amino-4- (2,4-dichlorophenyl) butyric acid; (S) -3-amino -4- (2-Chlorophenyl) -butyric acid; (S) -3-amino-4- (2-cyanophenyl) -butyric acid; (S) -3-amino-4- (2-fluorophenyl) -butyric acid; S) -3-amino-4- (2-furyl) -butyric acid; (S) -3-amino-4- (2-methylphenyl) -butyric acid; (S) -3-amino-4- (2-naphthyl) ) -Butyric acid; (S) -3-amino-4- (2-thienyl) -butyric acid; (S) -3-amino-4- (2-trifluoromethylphenyl) -butyric acid; (S) -3-amino -4- (3,4-dichlorophenyl) butyric acid; (S) -3-amino-4- (3,4-difluorophenyl) butyric acid; (S) -3-amino-4- (3-benzothienyl) -butyric acid (S) -3-amino-4- (3-chlorophenyl) -butyric acid; (S) -3-amino-4- (3-cyanophenyl) -butyric acid; (S) -3-amino-4- (3) -Fluorophenyl) -butyric acid; (S) -3-amino-4- (3-methylphenyl) -butyric acid; (S) -3-amino-4- (3-pyridyl) -butyric acid; (S) -3- Amino-4- (3-thienyl) -butyric acid; (S) -3-amino-4- (3-trifluoromethylphenyl) -butyric acid; (S) -3-amino-4- (4-bromophenyl)- Butyric acid; (S) -3-amino-4- (4-chlorophenyl) butyric acid; (S) -3-amino-4- (4-cyanophenyl) -butyric acid; (S) -3-amino-4- (4) -Fluorophenyl) Butyric acid; (S) -3-amino-4- (4-iodophenyl) -butyric acid; (S) -3-amino-4- (4-methylphenyl) -butyric acid; (S) -3- Amino-4- (4-nitrophenyl) -butyric acid; (S) -3-amino-4- (4-pyridyl) -butyric acid; (S) -3-amino-4- (4-trifluoromethylphenyl)- Butyric acid; (S) -3-amino-4-pentafluoro-phenylbutyric acid; (S) -3-amino-5-hexenoic acid; (S) -3-amino-5-hexic acid; (S) -3-hexinic acid Amino-5-phenylpentanoic acid; (S) -3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydro Pyridine-4-carboxylic acid; 3-amino-3- (2-chlorophenyl) -propionic acid; 3-amino-3- (2) -Thienyl) -propionic acid; 3-amino-3- (3-bromophenyl) -propionic acid; 3-amino-3- (4-chlorophenyl) -propionic acid; 3-amino-3- (4-methoxyphenyl) -Propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartate γ- Benzyl ester; L-β-homoglutamic acid δ-benzyl ester; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionin; L-β-homophenylalanine; L-β-homoproline; L-β -Homotryptophane; L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolicin; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β -Homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoaspartic acid; (R) -β-phenylalanine; L-β-homoaspartic acid γ-t-Butyl ester; L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolicin; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7 -Pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; Ot-butyl-L-β-homohydroxy-proline; Ot-butyl-L-β-homoseline; Ot-butyl- It includes, but is not limited to, L-β-homothreonine; Ot-butyl-L-β-homotyrosine; 2-aminocyclopentanecarboxylic acid; and 2-aminocyclohexanecarboxylic acid.

Amino acid analogs include analogs of alanine, valine, glycine, or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine are α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methyl-leucine; β- (1-naphthyl) -D- Alanine; β- (1-naphthyl) -L-alanine; β- (2-naphthyl) -D-alanine; β- (2-naphthyl) -L-alanine; β- (2-pyridyl) -D-alanine; β- (2-pyridyl) -L-alanine; β- (2-thienyl) -D-alanine; β- (2-thienyl) -L-alanine; β- (3-benzothienyl) -D-alanine; β -(3-benzothienyl) -L-alanine; β- (3-pyridyl) -D-alanine; β- (3-pyridyl) -L-alanine; β- (4-pyridyl) -D-alanine; β- (4-Pyridyl) -L-alanine; β-chloro-L-alanine; β-cyano-L-alanine; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine; β-cyclopentene-1-yl- Alanine; β-cyclopentyl-alanine; β-cyclopropyl-L-Ala-OH. Dicyclohexylammonium salt; β-t-butyl-D-alanine; β-t-butyl-L-alanine; γ-aminobutyric acid; L-α, β-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2, 5-Dihydro-D-Phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,5-trifluoro-butyric acid; 3-fluoro-valine 4,4,5-Trifluoro-valine; 4,5-dehydro-L-leu-OH. Dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl- D-Gly-OH. Dicyclohexylammonium salt; cyclopentyl-Gly-OH. Dicyclohexylammonium salt; D-α, β-diaminopropionic acid; D-α-aminobutyric acid; D-α-t-butylglycine; D- (2-thienyl) glycine; D- (3-thienyl) glycine; D- 2-Aminocaproic acid; D-2-indanylglycine; D-allylglycine-dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyric acid; (2-bromo) Phenyl) glycine; (2-methoxyphenyl) glycine; (2-methylphenyl) glycine; (2-thiazoyl) glycine; (2-thienyl) glycine; 2-amino-3- (dimethylamino) -propionic acid; L- α, β-diaminopropionic acid; L-α-aminobutyric acid; L-α-t-butylglycine; L- (3-thienyl) glycine; L-2-amino-3- (dimethylamino) -propionic acid; L -2-Aminocaproate dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine. Dicyclohexylammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α, γ-diaminobutyric acid; L-α, γ-diamino Butyric acid; β-cyclopropyl-L-alanine; (N-β- (2,4-dinitrophenyl))-L-α, β-diaminopropionic acid; (N-β-1- (4,4-dimethyl-) 2,6-dioxocyclohexa-1-iriden) ethyl) -D-α, β-diaminopropionic acid; (N-β-1- (4,4-dimethyl-2,6-dioxocyclohexa-1) -Ilidene) Ethyl) -L-α, β-diaminopropionic acid; (N-β-4-methyltrityl) -L-α, β-diaminopropionic acid; (N-β-allyloxycarbonyl) -L-α , Β-diaminopropionic acid; (N-γ-1- (4,4-dimethyl-2,6-dioxocyclohexa-1-iriden) ethyl) -D-α, γ-diaminobutyric acid; (N-γ -1- (4,4-dimethyl-2,6-dioxocyclohexa-1-iriden) ethyl) -L-α, γ-diaminobutyric acid; (N-γ-4-methyltrityl) -D-α, γ-Diaminobutyric acid; (N-γ-4-methyltrityl) -L-α, γ-diaminobutyric acid; (N-γ-allyloxycarbonyl) -L-α, γ-diaminobutyric acid; D-α, γ- Diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid Includes, but is not limited to, L-allylglycine; L-homocyclohexylalanine; and N- (2-hydroxy-4-methoxy-Bzl) -Gly-OH.

Amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine are citrulin; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulin; Lys (Me) _2- OH; Lys. (N ₃ ) -OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptandic acid; L- Ornithine; (Nδ-1- (4,4-dimethyl-2,6-dioxo-cyclohexa-1-iriden) ethyl) -D-ornithine; (Nδ-1- (4,4-dimethyl-2,6-dioxo) -Cyclohexa-1-ylidene) ethyl) -L-ornithine; (Nδ-4-methyltrithine) -D-ornithine; (Nδ-4-methyltrithine) -L-ornithine; D-ornithine; L-ornithine; Arg ( Me) (Pbf) -OH; Arg (Me) _2- OH (asymmetric); Arg (Me) 2-OH (symmetric); Lys (ivDde) -OH; Lys (Me) 2-OH. It includes, but is not limited to, HCl; Lys (Me3) -OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogs include aspartic acid or glutamic acid analogs. Examples of amino acid analogs of aspartic acid and glutamic acid are α-methyl-D-glutamic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)- L-Glutamic Acid; [N-α- (4-Aminobenzoyl)]-L-Glutamic Acid; 2,6-Diaminopimeric Acid; L-α-Aminosveric Acid; D-2-Aminoadipic Acid; D-α-Aminosverin Acids; α-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; treo-β-methyl-asparaginic acid; γ-carboxy-D-glutamic acid γ, γ-di-t-butyl ester; γ-carboxy- It includes, but is not limited to, L-glutamic acid γ, γ-di-t-butyl ester; Glu (OAll) -OH; L-Asu (OtBu) -OH; and pyroglutamic acid.

Amino acid analogs include analogs of cysteine and methionine. Examples of amino acid analogs of cysteine and methionine are Cys (farnesyl) -OH, Cys (farnesyl) -OMe, α-methyl-methionine, Cys (2-hydroxyethyl) -OH, Cys (3-aminopropyl) -OH. , 2-Amino-4- (ethylthio) butyric acid, butionine, butionine sulfoxymine, etionine, methionine methylsulfonium chloride, selenomethionin, cysteine acid, [2- (4-pyridyl) ethyl] -DL-penicillamine, [2 -(4-Pyridyl) ethyl] -L-Cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine, benzyl -D-Cysteine, benzyl-L-Cysteine, benzyl-DL-Homocysteine, Carbamoyl-L-Cysteine, Carboxyethyl-L-Cysteine, Carboxymethyl-L-Cysteine, Diphenylmethyl-L-Cysteine, Ethyl-L-Cysteine , Methyl-L-Cysteine, t-Butyl-D-Cysteine, Trityl-L-Homocysteine, Trityl-D-Penisylamine, Sistathionine, Homocystine, L-Homocystine, (2-Aminoethyl) -L-Cysteine, Sereno-L -Includes, but is not limited to, cystine, cystathionine, Cys (StBu) -OH, and acetamide methyl-D-peniciramine.

Amino acid analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine are β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinolin-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2- (trifluoromethyl) -D-phenylalanine, 2- (trifluoromethyl) -L-phenylalanine, 2- Bromo-D-Phenylalanine, 2-Bromo-L-Phenylalanine, 2-Chloro-D-Phenylalanine, 2-Chloro-L-Phenylalanine, 2-Cyano-D-Phenylalanine, 2-Cyano-L-Phenylalanine, 2-Fluoro- D-Phenylalanine, 2-Fluoro-L-Phenylalanine, 2-Methyl-D-Phenylalanine, 2-Methyl-L-Phenylalanine, 2-Nitro-D-Phenylalanine, 2-Nitro-L-Phenylalanine, 2,4,5- Trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine , 3,4-Difluoro-D-Phenylalanine, 3,4-difluoro-L-Phenylalanine, 3,4-dihydroxy-L-Phenylalanine, 3,4-dimethoxy-L-Phenylalanine, 3,5,3'-triiodo- L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3- (trifluoromethyl) -D-phenylalanine, 3- (tri) Fluoromethyl) -L-Phenylalanine, 3-Amino-L-Tyrosine, 3-Bromo-D-Phenylalanine, 3-Bromo-L-Phenylalanine, 3-Chloro-D-Phenylalanine, 3-Chloro-L-Phenylalanine, 3- Chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D- Phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-fe Nylalanine, 3-Nitro-D-Phenylalanine, 3-Nitro-L-Phenylalanine, 3-Nitro-L-Tyrosine, 4- (Trifluoromethyl) -D-Phenylalanine, 4- (Trifluoromethyl) -L-Phenylalanine, 4-Amino-D-Phenylalanine, 4-Amino-L-Phenylalanine, 4-Benzoyl-D-Phenylalanine, 4-Benzoyl-L-Phenylalanine, 4-bis (2-chloroethyl) Amino-L-Phenylalanine, 4-Bromo- D-Phenylalanine, 4-Bromo-L-Phenylalanine, 4-Chloro-D-Phenylalanine, 4-Chloro-L-Phenylalanine, 4-Cyano-D-Phenylalanine, 4-Cyano-L-Phenylalanine, 4-Fluoro-D- Includes Phenylalanine, 4-Fluoro-L-Phenylalanine, 4-Iodo-D-Phenylalanine, 4-Iodo-L-Phenylalanine, Homophenylalanine, Syroxine, 3,3-Diphenylalanine, Sylonine, Ethyl-tyrosine, and Methyl-tyrosine ..

Amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline. Not limited to.

Amino acid analogs include serine and threonine analogs. Examples of amino acid analogs of serine and threonine are 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2 −Amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3 Includes, but is not limited to, −ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and α-methylserine.

Amino acid analogs include tryptophan analogs. Examples of amino acid analogs of tryptophan are α-methyl-tryptophan; β- (3-benzothienyl) -D-alanine; β- (3-benzothienyl) -L-alanine; 1-methyl-tryptophan; 4-methyl. -Tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5- Methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan 7-Bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norhalman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorhalman-1- Carboic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norhalman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan. Not limited.

In some embodiments, the amino acid analog is racemic. In some embodiments, the D isomer of the amino acid analog is used. In some embodiments, the L isomer of the amino acid analog is used. In other embodiments, the amino acid analog comprises a chiral center within the R or S configuration. In yet another embodiment, the amino group of the β-amino acid analog is replaced with a protecting group such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl and the like. In yet another embodiment, the carboxylic acid functional group of the β-amino acid analog is protected, for example, as an ester derivative. In some embodiments, salts of amino acid analogs are used.

A "non-essential" amino acid residue is a wild-type polypeptide without abolishing or substantially altering its essential biological or biochemical activity (eg, receptor binding or activation). It is a residue that can be changed from the type sequence. An "essential" amino acid residue is a residue that, when varied from the wild-type sequence of a polypeptide, results in the loss or substantial loss of the essential biological or biochemical activity of the polypeptide.

A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, families of amino acid residues with similar side chains are defined. These families include amino acids with basic side chains (eg K, R, H), amino acids with acidic side chains (eg D, E), and uncharged polar side chains (eg G, N, Q). , S, T, Y, C), amino acids with non-polar side chains (eg, A, V, L, I, P, F, M, W), beta-branched side chains (eg, eg Includes amino acids with T, V, I) and amino acids with aromatic side chains (eg, Y, F, W, H). Thus, the predicted non-essential amino acid residues within the polypeptide are replaced, for example, with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on equal volume ratio considerations (eg, norleucine to methionine) or other properties (eg, 2-thienylalanine to phenylalanine, or 6-Cl-tryptophan to tryptophan). be.

B. Detailed Description Molecular cloning and characterization of Wnt3a is described by Saitoh et al., Biochem Biophys Res Commun, 2001, 284 (5): 1168-75. Natural human Wnt3a is a lipid-modified growth factor consisting of 352 amino acids (SEQ ID NO: 1) and is an effective growth factor in activating stem cells or stimulating their self-renewal. In some cases, due to their hydrophobic nature, active Wnt3a polypeptides or other Wnt polypeptides are difficult to purify to homogeneity at scalable levels. In one aspect of the specification, a method for purifying a Wnt polypeptide is described. In some cases, purification methods improve the purity and yield of human Wnt polypeptides. In some embodiments, the purified Wnt polypeptide is used as part of a therapeutic regimen. In some embodiments, the purified Wnt polypeptide is used as a therapeutic polypeptide.

As described elsewhere herein, Wnt polypeptides form a family of highly conserved secretory signaling molecules. The Wnt polypeptide can be a mammalian Wnt polypeptide, such as a human Wnt polypeptide. In some embodiments, the Wnt polypeptide is a Wnt1 polypeptide sequence, a Wnt2 polypeptide sequence, a Wnt2b (or Wnt13) polypeptide sequence, a Wnt3 polypeptide sequence, a Wnt3a polypeptide sequence, a Wnt4 polypeptide sequence, a Wnt5a polypeptide sequence. , Wnt5b polypeptide sequence, Wnt6 polypeptide sequence, Wnt7a polypeptide sequence, Wnt7b polypeptide sequence, Wnt8a polypeptide sequence, Wnt8b polypeptide sequence, Wnt9a (Wnt14, or Wnt14b) polypeptide sequence, Wnt9b (Wnt14b, or Wnt15) poly Includes peptide sequences, Wnt10a polypeptide sequences, Wnt10b (or Wnt12) polypeptide sequences, Wnt11 polypeptide sequences, Wnt-16a polypeptide sequences, or Wnt-16b polypeptide sequences, or functionally active variants thereof. In some embodiments, the Wnt polypeptide is selected from the group consisting of Wnt3a polypeptide, Wnt5a polypeptide, Wnt10b polypeptide, and functionally active Wnt variants thereof. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide or a functionally active variant thereof. In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide or a functionally active variant thereof. In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide or a functionally active variant thereof. In some embodiments, the Wnt3a polypeptide is a human Wnt3a polypeptide or a functionally active variant thereof. In some cases, the sequence identity of the Wnt3a polypeptide of the functionally active variant is at least about 30% relative to the native Wnt3a polypeptide sequence, such as the human Wnt3a polypeptide sequence (eg, SEQ ID NO: 1). About 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some cases, the sequence identity of the Wnt3a polypeptide of the functionally active variant is at most about 30% relative to the native Wnt3a polypeptide sequence, such as the human Wnt3a polypeptide sequence (eg, SEQ ID NO: 1). About 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. Optionally, the amino acid sequence of the Wnt3a polypeptide is set forth in SEQ ID NO: 1, or the Wnt3a polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 2.

In some cases, the purification schemes described herein are simple and inexpensive. In some cases, the purification schemes described herein allow purification of the functionally active form of the Wnt polypeptide with high purity. In some embodiments, the purification methods described herein allow the purification of Wnt polypeptides from the culture medium of recombinant host cells. In some cases, the recombinant host cell or expression cell line may be a prokaryotic host cell or prokaryotic cell line, a yeast host cell or yeast-expressing cell line, an insect host cell or insect-expressing cell line, or a mammalian host cell or mammalian-expressing cell. It is a system.

In some embodiments, the recombinant host cell or expressing cell line is a yeast host cell or yeast expressing cell line. Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains such as INVSc1.

In some embodiments, the recombinant host cell or expressing cell line is an insect host cell or insect expressing cell line. Exemplary insect cell lines include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five ™ cells, and expressSF + ™ cells.

In some embodiments, the recombinant host cell or expressing cell line is a mammalian host cell or mammalian expressing cell line. In some cases, a mammalian cell line is a cell that has the ability to integrate a stable cell line, or genetic material of interest, into its own genome and express the product of the genetic material after generations of cell division. It is a system. In some embodiments, the mammalian cell line is a cell line that is compatible with the Pharmaceutical Manufacturing and Quality Control Standards (cGMP). Exemplary mammalian cell lines are the 293A cell line, the 293FT cell line, the 293F cell line, the 293H cell line, the CHO DG44 cell line, the CHO-S cell line, the CHO-K1 cell line, the Expi293F ™ cell line, and the Flp-In ™ cell line. -REx ™ 293 cell line, Flp-In ™ -293 cell line, Flp-In ™ -3T3 cell line, Flp-In ™ -BHK cell line, Flp-In ™ -CHO Cell line, Flp-In ™ -CV-1 cell line, Flp-In ™ -Jurkat cell line, FreeStyle 2933-F cell, FreeStyle CHO-S cell, GripTite 293MSR Cell line, GS-CHO cell line, HepaRG ™ cell, T-REx ™ Jurkat cell line, Per. Includes, but is not limited to, C6 cells, T-REx ™ -293 cell line, T-REx ™ -CHO cell line, and T-REx ™ -HeLa cell line. In some embodiments, the mammalian cell line is a CHO cell line. In some embodiments, the mammalian cell line is a CHO-S cell line. As used herein, "CHO cell" or "CHO cell line" is a generic term that includes different types of CHO cells and CHO cell lines.

In some embodiments, mammalian host cells are grown as a liquid suspension culture in soft agar, on top of soft agar, or as a monolayer. In some embodiments, CHO cells are grown as a liquid suspension culture in soft agar, on top of soft agar, or as a monolayer. In some embodiments, CHO cells are cultured in a liquid suspension culture. In some cases, the culture medium contains serum. Non-limiting examples of sera include fetal bovine serum (FBS), HyClone Fetal Clone II and HyClone Fetal Clone III, iron-supplemented fetal bovine serum (ICS), and human platelet lysates. In some embodiments, the serum is FBS. In some embodiments, the FBS present in the medium is at most about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8 %, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or less. In some embodiments, the FBS present in the medium is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. , 9%, 10%, 11%, 12%, 13%, 14%, 15%, or more.

In some embodiments, serum (eg, FBS) is required to induce expression and secrete the Wnt polypeptide (eg, Wnt3a polypeptide) into the culture medium. In some embodiments, serum supplies one or more essential factors used for lipid modification on Wnt polypeptides (eg, residues such as Ser209 in Wnt3a). In some embodiments, serum is used to transport the Wnt polypeptide (eg, Wnt3a) from the endoplasmic reticulum (ER) to the Golgi apparatus and then to the cell membrane, with one or more chaperone proteins and a parent. Supply oily molecules.

In some embodiments, the serum is modified to remove the lipid component. In some cases, the defined set of lipids is introduced into the modified serum. Optionally, a stripper such as charcoal is used to remove non-polar lipophilic materials (eg, viruses, growth factors, and hormones) from the serum. Optionally, a lipid supplement is introduced into the modified serum. Non-limiting examples of lipid supplements include Lipid Mixture 1 (Sigma-Aldrich), Lipid Mixture 2 (Sigma-Aldrich), Lipogro® (Rocky Mountain Biologics), and Chemical Systems.

In some embodiments, mammalian host cells (eg, CHO cells or CHO-S cells) are cultured in serum-free medium. Non-limiting examples of serum-free media are CD CHO medium, CD CHO AGT ™ medium, CD OptiCHO ™ medium, CHO-S-SFM.
II (optionally containing hypoxanthin and thymidin), CD 293 AGT ™ medium, adenovirus expression medium (AEM), FreeStyle 293 expression medium, EX-CELL® 302 serum-free medium, EX-CELL® 325 PF CHO serum-free medium, EX-CELL® CD CHO-2 animal component-free medium, EX-CELL® CD CHO-3 medium, and EX-CELL® Includes CDHO DHFR ^- animal component-free medium.

In some embodiments, the serum-free medium contains one or more additional supplements. In some embodiments, the additional supplement is serum. In some cases, the serum is FBS. In some cases, the FBS present in the serum-free medium is at most about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or less. In some cases, the FBS present in the serum-free medium is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 %, 10%, 11%, 12%, 13%, 14%, 15%, or more.

In some embodiments, the additional supplement is a lipid supplement. Non-limiting examples of lipid supplements include Lipid Mixture 1 (Sigma-Aldrich), Lipid Mixture 2 (Sigma-Aldrich), Lipogro® (Rocky Mountain Biologics), and Chemical Systems. In some embodiments, the serum-free medium contains a lipid supplement.

In some embodiments, the additional supplement is a serum substitute. In some embodiments, the serum alternatives are EXCYTE (Millipore), EX-CYTE + human serum albumin + AOF ITS (insulin, transferrin, and selenium; Millipore), human serum albumin (Millipore), heparin sulfate (Sigma), the present invention. It is selected from lipids such as, but not limited to, DMPC and cholesterol, Cell-ess (Essential Pharmaceuticals), which are described elsewhere in the specification. Optionally, the Wnt polypeptide in the presence of a serum substitute is secreted into the conditioned medium. In some cases, Wnt polypeptides in the presence of serum substitutes are not secreted into the conditioned medium. Optionally, the Wnt3a polypeptide in the presence of a serum substitute is secreted into the conditioned medium. In some cases, the Wnt3a polypeptide in the presence of serum substitutes is not secreted into the conditioned medium.

In some embodiments, expression vectors that tolerate serum-free medium conditions are used. In some cases, the expression vector results in the secretion of a high copy number of the desired transcript and the polypeptide of interest. Optionally, the expression vector is compatible with a mammalian cell line compatible with cGMP. Non-limiting examples of mammalian expression vectors include pOptivec vector, pTagT ™ vector, BacMam pCMV-Dest vector, Flp-In ™ core system, Gateway ™ series vector, HaloTag ™. Includes vectors, Flexi® vectors, pCMVTNT® vectors, and pcDNA ™ 4 / TO vectors. In some embodiments, the expression vector is selected from the pOptivec vector and the pTageT ™ vector. The optivec vector is a TOPO® adaptive bicistronic plasmid that allows rapid cloning of genes containing secretory signals to mammals and genes of interest downstream of the CMV promoter. Dihydrofolate reductase selectable markers allow for rapid selection. Optionally, this vector is used for transient transfection of CHO-S cells. Optionally, the pTageT ™ vector is used for transient transfection of CHO-S cells and the creation of a stable cell line expressing the Wnt polypeptide (eg, Wnt3a).

In some embodiments, acidification of culture conditions is used to aid in Wnt secretion. In some cases, acidification of culture conditions is used to aid Wnt3a secretion. In some cases, acidification mimics the acidification within the vesicles. In some cases, acidification is cytoplasmic acidification. In some cases, acidification occurs naturally, such as acids secreted by cells growing in conditioned medium, or artificially, such as by the addition of acids such as acetic acid. In some cases, acidification aids in the secretion of Wnt polypeptides. In some cases, acidification aids in the secretion of Wnt3a polypeptides.

In some embodiments, the expression method of the Wnt polypeptide in yeast cells, insect cells, or mammalian cells follows a protocol well known in the art. Optionally, the Wnt polypeptide is expressed in mammalian cells. In some embodiments, a transcription inducer is used to induce the expression of the Wnt polypeptide. In some cases, transcription inducers include doxycycline, tetracycline, and cumermycin. In some cases, expression is constitutive or the polypeptide is expressed without an inducer. In some embodiments, the Wnt polypeptide is secreted into the medium. Optionally, conditioned medium is harvested and additional agents are added to solubilize the Wnt polypeptide. In some cases, the additional agent is a surfactant. In some cases, the surfactant is a nonionic surfactant, an anionic surfactant, or a zwitterionic surfactant. Exemplary surfactants are Brij-35, Brij-58, 3-[(3-colamidpropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 3-([3-colamidpropyl] dimethyl]. Ammonio) -2-hydroxy-1-propanesulfonate (CHASPO), nonyl phenoxypolethoxylethanol (NP-40), octyl glucoside, octyl thioglucoside, sodium dodecyl sulfate (SDS), Triton
Includes, but is not limited to, X-100, Triton X-114, Tween 20, and Tween 80. In some embodiments, the surfactant is CHAPS or Triton X-100. In some embodiments, the surfactant is CHAPS. CHAPS is a zwitterionic surfactant useful for solubilizing membrane proteins. In some embodiments, the Wnt polypeptide is also post-translational modified via glycosylation and palmitoylation.

Palmitoylation is a covalent bond of fatty acids such as palmitic acid to cysteine residues, serine residues, and threonine residues. In some cases, palmitoylation enhances the hydrophobicity of proteins and contributes to their association with membranes. For example, Nusse et al. S-palmitoylated mouse Wnt3a protein at a conserved cysteine residue (C77) (Willert et al., Nature, 2003, 423: 448-452) and palmitoylated Cys77. It has been reported that the mutant form of the mouse Wnt3a protein substituted with alanine (C77A) shows diminished ability to activate Wnt signaling but is normally secreted into culture medium. In addition, studies have shown that palmitoylation of Cys77 residues is important for biological activity.

In some embodiments, the Wnt polypeptide is palmitoylated at cysteine residues, serine residues, and / or threonine residues. In some embodiments, the Wnt3a polypeptide is palmitoylated at cysteine residues, serine residues, and / or threonine residues. Optionally, the Wnt3a polypeptide is palmitoylated at C77, S139, S181, S209, or S211 shown in SEQ ID NO: 1. Optionally, the Wnt3a polypeptide is palmitoylated at one or more of the amino acid positions selected from C77, S139, S181, S209, or S211 shown in SEQ ID NO: 1. Optionally, the Wnt3a polypeptide is palmitoylated at C77 set forth in SEQ ID NO: 1. Optionally, the Wnt3a polypeptide is palmitoylated at S209 set forth in SEQ ID NO: 1. Optionally, the Wnt3a polypeptide is palmitoylated at C77 and S209 set forth in SEQ ID NO: 1. In some cases, the Wnt3a protein is not palmitoylated at C77 set forth in SEQ ID NO: 1. In some cases, the Wnt3a protein is palmitoylated in S209 set forth in SEQ ID NO: 1 but not in C77 set forth in SEQ ID NO: 1.

In some embodiments, the Wnt polypeptide containing the palmitoylated residue is a functionally active mutant polypeptide, such as a functionally active variant of a Wnt3a native polypeptide (eg, human Wnt3a). Is. In some embodiments, the Wnt3a functionally active mutant polypeptide comprises a palmitoylated residue selected from C77, S139, S181, S209, and S211 shown in SEQ ID NO: 1. In some embodiments, the Wnt3a functionally active mutant polypeptide has a palmitoylated residue in S139 set forth in SEQ ID NO: 1. In some embodiments, the functionally active mutant Wnt3a polypeptide has a palmitoylated residue in S181 set forth in SEQ ID NO: 1. In some embodiments, the functionally active mutant Wnt3a polypeptide has a palmitoylated residue in S209 set forth in SEQ ID NO: 1. In some embodiments, the functionally active mutant Wnt3a polypeptide has a palmitoylated residue in S211 set forth in SEQ ID NO: 1. In some embodiments, the functionally active mutant Wnt3a polypeptide has palmitoylated residues at S209 and one other residue set forth in SEQ ID NO: 1. In some embodiments, the other residue does not contain C77. In some embodiments, the functionally active mutant Wnt3a polypeptide is palmitoylated in S209 set forth in SEQ ID NO: 1 but not in C77 set forth in SEQ ID NO: 1.

In some embodiments, the purification schemes described herein are Wnt1 polypeptide, Wnt2 polypeptide, Wnt2b (or Wnt13) polypeptide, Wnt3 polypeptide, Wnt3a polypeptide, Wnt4 polypeptide, Wnt5a polypeptide, Wnt5b polypeptide, Wnt6 polypeptide, Wnt7a polypeptide, Wnt7b polypeptide, Wnt8a polypeptide, Wnt8b polypeptide, Wnt9a (Wnt14 or Wnt14b) polypeptide, Wnt9b (Wnt14b or Wnt15) polypeptide, Wnt10a polypeptide, Wnt10b ( Or Wnt12) relating to a Wnt polypeptide selected from a polypeptide, a Wnt11 polypeptide, a Wnt-16a polypeptide, and a Wnt-16b polypeptide. Optionally, the purification schemes described herein relate to Wnt3a, Wnt5a, or Wnt10b polypeptides. Optionally, the purification schemes described herein relate to Wnt3a polypeptides. Optionally, the purification schemes described herein relate to Wnt5a polypeptides. Optionally, the purification schemes described herein relate to Wnt10b polypeptides.

In some embodiments, the purification scheme described herein corresponds to a cysteine residue corresponding to cysteine at amino acid position 77 set forth in SEQ ID NO: 1 or serine at amino acid position 209 set forth in SEQ ID NO: 1. With respect to the Wnt3a polypeptide having palmitoylation at the serine residue. In some embodiments, the purification scheme described herein selects a Wnt3a polypeptide having palmitoylation at the cysteine residue corresponding to the cysteine at position 77 of amino acid set forth in SEQ ID NO: 1. In some embodiments, the purification scheme described herein selects a Wnt3a polypeptide having palmitoylation at the serine residue corresponding to the serine at amino acid position 209 set forth in SEQ ID NO: 1. In some embodiments, the purification scheme described herein is a palmitoyl at the serine residue corresponding to the serine at amino acid position 209 set forth in SEQ ID NO: 1 and at least one other palmitoylated residue. Select a Wnt3a polypeptide that has a chemistry. In some embodiments, at least one other palmitoylated residue is not C77 shown in SEQ ID NO: 1. In some embodiments, the purification schemes described herein do not select Wnt3a polypeptides that have palmitoylation at the cysteine residue corresponding to the cysteine at amino acid position 77 set forth in SEQ ID NO: 1.

In some embodiments, the purification scheme described herein utilizes a separation step. In some embodiments, the separation step is an ion exchange purification step, a hydrophobic purification step, or an affinity purification step. In some cases, the separation step is an ion exchange purification step. Optionally, the ion exchange purification step utilizes beads immobilized with one or more sulfonated polycyclic aromatic compounds. Optionally, the ion exchange purification step utilizes a column on which one or more sulfonated polycyclic aromatic compounds are immobilized. A non-limiting example of a sulfonated polycyclic aromatic compound is Cibacron blue F3GA. In some cases, Cibacron blue F3GA is a triazinyl dye. Optionally, the ion exchange purification step uses beads and / or columns with triadinyl pigments immobilized. A non-limiting example of a chromatographic column on which Cibacron blue F3GA is immobilized is a Blue Sepharose column.

In some embodiments, purification is performed in batch mode with the use of beads immobilized with sulfonated polycyclic aromatic compounds. Generally, the Wnt polypeptide binds the sulfonated polycyclic aromatic compound to the immobilized beads in a binding buffer containing a low concentration of salt. High concentrations of salt destabilize the non-covalent ionic interaction between the protein and the beads, thereby allowing the Wnt polypeptide to elute. In some embodiments, the concentration of salt used in the binding buffer is at most 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50 mM, or less. In some embodiments, the concentration of salt used in the binding buffer is at least 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50 mM, or greater. In some embodiments, one or more wash buffers are used to remove unbound impurities. In some embodiments, at most one, two, three, four, five, or more cleaning steps are used. In some embodiments, at least one, two, three, four, five, or less cleaning steps are used. In some embodiments, the concentration of salt used in the wash buffer is at least 30, 40, 50, 60, 70, 80, 90, 100 mM, or greater. In some embodiments, the concentration of salt used in the wash buffer is at most 30, 40, 50, 60, 70, 80, 90, 100 mM, or less. In some embodiments, one or more elution steps follow. In some embodiments, the concentration of salt in the elution buffer is at least 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000 mM or more. In some embodiments, the concentration of salt in the elution buffer is at most 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350. , 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000 mM, or less. Exemplary salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, calcium phosphate, potassium phosphate, magnesium phosphate, sodium phosphate, ammonium sulfate, ammonium chloride, ammonium phosphate and the like. In some embodiments, surfactants are also formulated in binding buffer, wash buffer, and / or elution buffer. In some embodiments, the surfactant is CHAPS or Triton X-100. In some embodiments, the percentages of CHAPS or Triton X-100 are at least 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3 %, 3.5%, 4%, 4.5%, 5%, or more. In some embodiments, the percentages of CHAPS or Triton X-100 are at most 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or less. In some cases, tris (hydroxymethyl) methylamine HCl (Tris-HCl), 3-{[tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPS), N, N-bis (2-hydroxyethyl) glycine ( Bicin), N-tris (hydroxymethyl) methylglycine (tricin), 3- [N-tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid (TAPSO), 4--2-hydroxyethyl-1-piperazin Ethan Sulfonic Acid (HEPES), 3- (N-Morphorino) Propane Sulfonic Acid (MOPS), Piperazin-N, N'-Bis (2-Ethan Sulfonic Acid) (PIPES), 2- (N-Morphorino) Ethan Sulfonic Acid Use a buffer component such as (MES). In some cases, the pH of the buffer is at least 4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9 or higher. In some cases, the pH of the buffer is at most 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or less. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide.

In some embodiments, purification is performed using a column immobilized with sulfonated polycyclic aromatic compounds. Generally, the Wnt polypeptide binds the sulfonated polycyclic aromatic compound to an immobilized column in a binding buffer containing a low concentration of salt. High concentrations of salt destabilize the non-covalent ionic interaction between the polypeptide and the column beads, thereby allowing the Wnt polypeptide to elute. In some embodiments, the concentration of salt used in the binding buffer is at most 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50 mM, or less. In some embodiments, the concentration of salt used in the binding buffer is at least 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50 mM, or greater. In some embodiments, one or more wash buffers are used to remove unbound impurities. In some embodiments, at most one, two, three, four, five, or more cleaning steps are used. In some embodiments, at least one, two, three, four, five, or less cleaning steps are used. In some embodiments, the concentration of salt used in the wash buffer is at least 30, 40, 50, 60, 70, 80, 90, 100 mM, or greater. In some embodiments, the concentration of salt used in the wash buffer is at most 30, 40, 50, 60, 70, 80, 90, 100 mM, or less. In some embodiments, one or more elution steps follow. In some embodiments, the concentration of salt in the elution buffer is at least 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000 mM or more. In some embodiments, the concentration of salt in the elution buffer is at most 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350. , 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000 mM, or less. Exemplary salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, calcium phosphate, potassium phosphate, magnesium phosphate, sodium phosphate, ammonium sulfate, ammonium chloride, ammonium phosphate and the like. In some embodiments, surfactants are also formulated in binding buffer, wash buffer, and / or elution buffer. In some embodiments, the surfactant is CHAPS or Triton X-100. In some embodiments, the percentages of CHAPS or Triton X-100 are at least 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3 %, 3.5%, 4%, 4.5%, 5%, or more. In some embodiments, the percentages of CHAPS or Triton X-100 are at most 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or less. In some cases, tris (hydroxymethyl) methylamine HCl (tris-HCl), 3-{[tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPS), N, N-bis (2-hydroxyethyl) glycine ( Bicin), N-tris (hydroxymethyl) methylglycine (tricin), 3- [N-tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid (TAPSO), 4--2-hydroxyethyl-1-piperazin Ethan Sulfonic Acid (HEPES), 3- (N-Morphorino) Propane Sulfonic Acid (MOPS), Piperazin-N, N'-Bis (2-Ethan Sulfonic Acid) (PIPES), 2- (N-Morphorino) Ethan Sulfonic Acid Use a buffer component such as (MES). In some cases, the pH of the buffer is at least 4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9 or higher. In some cases, the pH of the buffer is at most 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or less. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide.

In some embodiments, the separation step is a hydrophobic purification step. In some embodiments, the hydrophobic purification step utilizes beads with an immobilized hydrophobic ligand, or a column with an immobilized hydrophobic ligand. Non-limiting examples of ligands include butyl, octyl, phenyl, protein A and the like.

In some embodiments, the separation step is an affinity purification step. In some embodiments, the affinity purification step utilizes beads with an immobilized affinity ligand or a column with an immobilized affinity ligand. Exemplary ligands include Frizzled receptor (Fzd) or fragments thereof, and low density lipoprotein receptor-related protein 6 (LRP6) or fragments thereof.

In some embodiments, the concentration and yield of the eluted Wnt polypeptide is measured prior to further purification steps. In some embodiments, the yield is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the yield is at most about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less. In some embodiments, the purity is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater. In some embodiments, the purity is at most about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less.

In some embodiments, the purified Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1 is purified by a separation step. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 is purified by a separation step. In some embodiments, a Wnt3a polypeptide comprising palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 and palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1. , Purify by separation step. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1 and palmitoylation at at least one other amino acid residue is purified by a separation step. .. In some embodiments, at least one other amino acid residue is not C77 set forth in SEQ ID NO: 1.

In some embodiments, the concentration of the Wnt3a molecular species containing palmitoylation of serine 209 in the eluted product is greater than the concentration of the Wnt3a molecular species containing palmitoylation of cysteine 77. In some embodiments, the concentrations of the two Wnt3a molecular species are defined by values such as ratios. In some embodiments, the ratio of Wnt3a serine 209 to Wnt3a cysteine 77 molecule is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, Between about 75:25, about 80:20, about 85:15, about 90:10, about 95: 5, about 99: 1, or about 100: 0. In some embodiments, the concentration of the Wnt3a molecular species containing palmitoylation of serine 209 in the eluted product is greater than the concentration of the Wnt3a molecular species containing palmitoylation of cysteine 77 and serine 209. In some embodiments, the concentrations of the two Wnt3a molecular species are defined by values such as ratios. In some embodiments, the ratio of Wnt3a serine 209 to cysteine 77 / serine 209 is about 50:50, about 55:45, about 60:40, about 65:35, about 70. Between: 30, about 75:25, about 80:20, about 85:15, about 90:10, about 95: 5, about 99: 1, or about 100: 0.

In some embodiments, the further purification step is to utilize prefabricated liposomes. In some embodiments, liposomes eliminate serum (FBS) from the polypeptide preparation. In some embodiments, this latter step takes advantage of the hydrophobicity of the Wnt polypeptide and further includes, for example, a subsequent gel filtration purification step and / or a chromatographic purification step (eg, heparin sulfate immobilized column). Eliminates the need for purification steps or subsequent purification steps.

In some embodiments, liposomes are made using methods well known in the art. Liposomes are artificially prepared spherical vesicles that make up the lipid bilayer and aqueous core of the lamellar phase. There are multiple types of liposomes, including multimembrane vesicles (MLVs), small monolayer liposome vesicles (SUVs), large monolayer vesicles (LUVs), and spiral vesicles. In some cases, liposomes are formed by phospholipids. In some embodiments, the phospholipid is divided into a phospholipid with a diacylglyceride structure, or a phospholipid derived from a phosphosphingolipid. In some embodiments, the diacylglyceride structures are phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lesitin) (PC), phosphatidylserine (PS), and phosphatidylinositol. (PI), phosphatidyl inositol phosphate (PIP), phosphatidyl inositol bisphosphate (PIP2), and phosphoinositides such as phosphatidyl inositol triphosphate (PIP3). In some embodiments, the phosphosphingolipid comprises a ceramide phosphorylcholine, a ceramide phosphorylethanolamine, and a ceramide phosphoryl lipid. In some embodiments, the liposomes are formed from phosphatidylcholine.

In some embodiments, the lipid is also _{selected based on its phase transition temperature (Tm} ), or the interface temperature between the liquid crystal phase and the gel phase. In some embodiments, _Tm is governed by the molecular species of the head group, the length of the hydrocarbone, the degree of unsaturation, and the charge. For example, short chain lipids (lipids containing 8, 10, or 12 tail carbon chain lengths) have a liquid crystal phase at temperatures below 4 ° C. However, liposomes made from these short-chain carbon lipids are toxic to cells because they lyse cell membranes. Liposomes made from lipids with longer carbon chains are not toxic to cells, but their transition temperature is high. _{For example, the T m} of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), which has a tail carbon length of 16, is about 41 ° C. In some embodiments, _{T m} of the lipids used herein, about 10 ° C. ~ about 37 ° C., 15 ° C. ~ about 30 ° C., 18 ° C. ~ about 27 ° C., or 21 ° C. ~ about 25 ° C. Between. In some embodiments, T _m of the lipids used herein is at least 22 ° C., 23 ° C., 24 ° C., or greater than this. In some embodiments, T _m of the lipids used herein, also 22 ° C. most, 23 ° C., a 24 ° C., or less than this. In some embodiments, the length of the tail carbon of the lipids used herein is at least about 12, 13, 14, or more. In some embodiments, the length of the tail carbon of the lipids used herein is at most about 12, 13, 14 or less.

In some embodiments, lipids are further selected based on the net charge of the liposome. In some embodiments, the pH is about 4.0 to about 10.0, about 5.0 to about 9.0, about 6.5 to about 8.0, about 7.0 to about 7.8, or about. The net charge of liposomes between 7.2 and about 7.6 is zero. In some embodiments, the net charge of the liposome at pH about 7.3, about 7.4, or about 7.5 is zero. In some embodiments, the pH is about 4.0 to about 10.0, about 5.0 to about 9.0, about 6.5 to about 8.0, about 7.0 to about 7.8, or about. The net charge of liposomes between 7.2 and about 7.6 is positive. In some embodiments, the net charge of the liposome at pH about 7.3, about 7.4, or about 7.5 is positive. In some embodiments, the pH is about 4.0 to about 10.0, about 5.0 to about 9.0, about 6.5 to about 8.0, about 7.0 to about 7.8, or about. The net charge of liposomes between 7.2 and about 7.6 is negative. In some embodiments, the net charge of liposomes at pH of about 7.3, about 7.4, or about 7.5 is negative.

In some embodiments, the lipids are 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-tetradeca. Noyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (MPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serin (DMPS), and 1,2-dihexanoyl-sn- It is selected from glycero-3-phosphocholine (DMPG). In some embodiments, the lipid is DMPC.

In some embodiments, additional lipids are made into liposomes. In some embodiments, the additional lipid is cholesterol. In some cases, the concentration of phosphatidylcholine, such as DMPC and cholesterol, is defined by a value such as ratio. In some embodiments, the ratio of concentrations of phosphatidylcholine, such as DMPC and cholesterol, is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about. It is between 80:20, about 85:15, about 90:10, about 95: 5, about 99: 1, or about 100: 0. In some embodiments, the ratio of concentrations of phosphatidylcholine, such as DMPC and cholesterol, is about 90:10. In some embodiments, the concentration unit is molar. In some embodiments, the ratio is mol: mol.

In some embodiments, the Wnt polypeptide is at least about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0. Reconstitute with liposomes at concentrations of 25, 0.3, 0.4, 0.5 ng / μL, or greater. In some embodiments, the Wnt polypeptide is at most about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05. , 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0 Reconstitute with liposomes at concentrations of .25, 0.3, 0.4, 0.5 ng / μL, or less. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide.

In some embodiments, the Wnt polypeptide is liposomal at a ratio of Wnt polypeptide to liposome, at least about 0.1:50, 0.5:30, 1:20, or 1:14, or greater. Reconstruct with. In some embodiments, the Wnt polypeptide is at most about 0.1:50, 0.5:30, 1:20, or 1:14, or less, in a Wnt polypeptide to liposome ratio. Reconstitute with liposomes. In some cases, the ratio is weight to weight ratio. In some cases, the unit of Wnt polypeptide is nanogram units.

In some embodiments, the temperature at which the Wnt polypeptide is reconstituted with the liposomes is at least between about 15 ° C. and about 37 ° C., about 18 ° C. and about 33 ° C., or about 20 ° C. and about 28 ° C. In some embodiments, the temperature is at least about 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, or higher. In some embodiments, the temperature is at most about 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, or less. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide.

In some embodiments, the Wnt polypeptide is incorporated into the liposome membrane. In some cases, the Wnt polypeptide projects from the liposome membrane to the surface of the lipid membrane. In some cases, the Wnt polypeptide is not integrated into the aqueous core of the liposome. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide is incorporated into the liposome membrane. In some cases, the Wnt3a polypeptide projects from the liposome membrane to the surface of the lipid membrane. In some cases, the Wnt3a polypeptide is not integrated into the aqueous core of the liposome.

In some embodiments, the Wnt polypeptide reconstituted with liposomes is referred to as the liposome Wnt polypeptide or L-Wnt. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the Wnt3a polypeptide reconstituted with the liposomes is referred to as the liposome Wnt3a polypeptide or L-Wnt3a. In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide. In some embodiments, the Wnt5a polypeptide reconstituted with the liposomes is referred to as the liposome Wnt5a polypeptide or L-Wnt5a. In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide. In some embodiments, the Wnt10b polypeptide reconstituted with the liposomes is referred to as the liposome Wnt10b polypeptide or L-Wnt10b.

In some embodiments, L-Wnt is subjected to a centrifugation step and then suspended in a buffer solution such as phosphate buffered saline (PBS). In some cases, L-Wnt is stored under nitrogen. In some cases, L-Wnt is stable under nitrogen with no substantial loss of activity. Optionally, L-Wnt is stored at a temperature between about 1 ° C and about 8 ° C. In some cases, L-Wnt is at least about 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 ° C., or higher, with no substantial loss of activity. Is stable. In some cases, L-Wnt is associated with a substantial loss of activity at temperatures of at most about 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 ° C., or less. It is stable without. In some embodiments, L-Wnt is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 356, 400, 700, It is stable for 1000 days, or beyond, with no substantial loss of activity. In some embodiments, L-Wnt is at most about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95. , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 356, 400, 700. Stable for 1000 days, or less, with no substantial loss of activity.

In some embodiments, L-Wnt3a is subjected to a centrifugation step and then suspended in a buffer solution such as phosphate buffered saline (PBS). In some cases, L-Wnt3a is stored under nitrogen. In some cases, L-Wnt3a is stable under nitrogen with no substantial loss of activity. Optionally, L-Wnt3a is stored at a temperature between about 1 ° C and about 8 ° C. In some cases, L-Wnt3a is at least about 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 ° C., or higher, with no substantial loss of activity. Is stable. In some cases, L-Wnt3a is associated with a substantial loss of activity at temperatures at most about 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 ° C., or less. It is stable without. In some embodiments, L-Wnt3a is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, It is stable for 190, 200, 300, 356, 400, 700, 1000 days, or more, without substantial loss of activity. In some embodiments, L-Wnt3a is at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180. , 190, 200, 300, 356, 400, 700, 1000 days, or less, stable without substantial loss of activity.

In some cases, the term "without substantial loss of activity" refers to the functional activity of a liposome Wnt polypeptide being close to that of the corresponding native Wnt polypeptide in the absence of liposomes. In some cases, the functional activity of the liposome Wnt polypeptide is at least about 100%, 99%, 95%, 90%, 85%, 80%, 75%, 70 as compared to the functional activity of the native Wnt polypeptide. %, 60%, 50%, 40%, or more. In some cases, the functional activity of the liposome Wnt polypeptide is at most about 100%, 99%, 95%, 90%, 85%, 80%, 75%, as compared to the functional activity of the native Wnt polypeptide. 70%, 60%, 50%, 40%, or less. In some cases, the functional activity of Wnt polypeptides can be described, for example, in mass spectrometry, assays associated with biomarker analysis described elsewhere herein, transplants such as subcapsular transplants, spinal fusion, Assays are used such as ALP staining, TRAP staining, and TUNEL staining, immunohistochemistry, and Micro-CT analysis and quantification for graft growth.

In some cases, the term "stable" refers to a Wnt polypeptide that is in a folded state and is not unfolded or degraded. In some cases, the term "stable" also refers to a Wnt polypeptide that retains functional activity without substantial loss of activity. In some cases, the assays used to determine stability are assays that establish the activity of Wnt polypeptides, such as the assays described above, and LSL cell-based assays, such as mouse LSL cell-based assays. Including.

In some embodiments, the purification methods described herein employ the hydrophobic domain of the Wnt polypeptide and do not interfere with the conformation or functional activity of the Wnt polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. Optionally, the hydrophobic domain of the Wnt3a polypeptide contains palmitoylation on the serine residue corresponding to serine 209 set forth in SEQ ID NO: 1.

In some embodiments, the affinity of the Wnt polypeptide for liposomes is smaller than the binding partner of the Wnt polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5b polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the affinity of the Wnt3a polypeptide for liposomes is smaller than the binding partner of the Wnt3a polypeptide. In some embodiments, the binding partner of the Wnt3a polypeptide comprises Frizzled protein, a fragment thereof, LRP6 protein, and a fragment thereof. In some embodiments, the affinity of Wnt3a for liposomes is between about 4 nM to about 50 nM, and about 4.5 nM to about 20 nM, and about 5 nM to about 15 nM. In some embodiments, the affinity of Wnt3a for liposomes is at least about 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, or more. In some embodiments, the affinity of Wnt3a for liposomes is at most about 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 11 nM, 12 nM. , 13 nM, 14 nM, or less. In some embodiments, the affinity of the Wnt3a polypeptide for its binding partner is between about 0.01 nM to about 4 nM, about 0.1 nM to about 4 nM, or about 1 nM to about 4 nM. In some cases, the affinity of the Wnt3a polypeptide for its binding partner is at least about 1.1 nM, 1.3 nM, 1.5 nM, 1.7 nM, 2 nM, 2.3 nM, 2.5 nM, 2.7 nM, 3 nM, 3. 1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, or more. In some cases, the affinity of the Wnt3a polypeptide for its binding partner is at most about 1.1 nM, 1.3 nM, 1.5 nM, 1.7 nM, 2 nM, 2.3 nM, 2.5 nM, 2.7 nM, 3 nM, 3 .1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, or less.

Different purification schemes result in different amounts of purified polypeptide. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 is in active form. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1 is in active form. In some embodiments, the Wnt3a polypeptide comprising palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1 and palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 , Active form. In some embodiments, the Wnt3a polypeptide comprising palmitoylation at the amino acid residue corresponding to serine 209 set forth in SEQ ID NO: 1 and palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 , Not in active form. In some embodiments, the Wnt3a polypeptide containing palmitoylation at the amino acid residue corresponding to cysteine 77 set forth in SEQ ID NO: 1 is not an active form. In some embodiments, active molecular species are identified using mass spectrometry coupled with additional assays to detect or measure the activity of Wnt polypeptides (eg, Wnt3a polypeptides). In some cases, additional assays include those associated with biomarker analysis described elsewhere herein, transplants such as subcapsular transplantation, spinal fusion, ALP staining, TRAP staining, and TUNEL staining. Includes immunohistochemistry, as well as Micro-CT analysis and quantification of graft growth.

In some embodiments, the concentration of the liposome Wnt3a molecular species containing palmitoylation of serine 209 in the eluted product is greater than the Wnt3a molecular species containing palmitoylation of cysteine 77. In some embodiments, the concentrations of the two Wnt3a molecular species are defined by values such as ratios. In some embodiments, the ratio of Wnt3a serine 209 to Wnt3a cysteine 77 molecule is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, Between about 75:25, about 80:20, about 85:15, about 90:10, about 95: 5, about 99: 1, or about 100: 0. In some embodiments, the concentration in the eluted product of the liposome Wnt3a molecular species containing palmitoylation of serine 209 is greater than that of the Wnt3a molecular species containing palmitoylation of cysteine 77 and serine 209. In some embodiments, the concentrations of the two Wnt3a molecular species are defined by values such as ratios. In some embodiments, the ratio of Wnt3a serine 209 to cysteine 77 / serine 209 is about 50:50, about 55:45, about 60:40, about 65:35, about 70. Between: 30, about 75:25, about 80:20, about 85:15, about 90:10, about 95: 5, about 99: 1, or about 100: 0.

In some cases, a mixture of two molecular species (eg, single modification / activity: double modification / inert) may be used according to the purification scheme described above, including the purification scheme described in the references disclosed herein. Is 1: 5) is obtained. Optionally, using the purification schemes described herein, the major polypeptide molecular species take a single modified (in Ser209), active configuration. Optionally, the purification methods described herein provide Wnt3a compositions that predominantly contain active forms of the polypeptide, including lipid modifications in Ser209. In some cases, the purification methods described herein provide a Wnt3a composition that is not contaminated with other polypeptide molecular species, such as the Cys77 Wnt3a molecular species.

In some embodiments herein, the method of preparing a liposome Wnt polypeptide comprises contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, and the amount of phospholipids constituting the liposome is about 10. Methods having a phase transition temperature of ° C. to about 25 ° C. are described. In some embodiments herein, a method of preparing a liposome Wnt polypeptide, comprising contacting a sample containing the Wnt polypeptide with an aqueous solution of the liposome, comprising the step of contacting the tail carbon of the phospholipids constituting the liposome. Methods are described in which the length is between about 12 carbons and about 14 carbons. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide.

In some embodiments herein, a method of preparing a liposome Wnt3a polypeptide, wherein (a) the Wnt3a polypeptide is taken from a conditioned medium containing Chinese hamster ovary (CHO) cells; (b). ) The step of introducing the Wnt3a polypeptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized; and (c) the step of eluting the Wnt3a polypeptide from the ion exchange column using a step gradient; d) A method comprising contacting the Wnt3a polypeptide with an aqueous solution of the liposome according to step (c) is disclosed.

In some embodiments herein, a method of preparing a liposome Wnt5a polypeptide, wherein (a) the Wnt5a polypeptide is taken from a conditioned medium containing Chinese hamster ovary (CHO) cells; (b). ) The step of introducing the Wnt5a polypeptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized; and (c) the step of eluting the Wnt5a polypeptide from the ion exchange column using a step gradient; d) A method comprising contacting the Wnt5a polypeptide with an aqueous solution of the liposome according to step (c) is disclosed.

In some embodiments herein, a method of preparing a liposome Wnt10b polypeptide, wherein (a) the Wnt10b polypeptide is taken from a conditioned medium containing Chinese hamster ovary (CHO) cells; (b). ) The step of introducing the Wnt10b polypeptide into an ion exchange column on which a sulfonated polycyclic aromatic compound is immobilized; and (c) the step of eluting the Wnt10b polypeptide from the ion exchange column using a step gradient; d) A method comprising contacting the Wnt10b polypeptide with an aqueous solution of the liposome according to step (c) is disclosed.

Functional assays that determine the biological activity of Wnt polypeptides are well known in the art, such as stabilizing β-catenin, promoting stem cell proliferation, and the amount of Wnt polypeptides present in non-functional assays. Quantification, such as immunostaining, ELISA, quantification on Coomassie-stained or silver-stained gels, and analysis of the ratio of functionally active Wnts to total Wnts may be included. An exemplary assay for biological activity involves contacting the Wnt composition with cells, eg, mouse L cells, and then for at least about 1 hour, for a time sufficient to stabilize β-catenin. Includes the step of culturing cells. The cells were then lysed and the cell lysates were resolved by SDS PAGE. The cell lysate component resolved by SDS PAGE was then transcribed into nitrocellulose and then probed with an antibody specific for β-catenin. Other assays for analyzing Wnt activity include transformation of C57MG and Xenopus animal cap assay for induction of target genes. See U.S. Pat. No. 7,335,643 for a further exemplary assay.

C. Usage The methods, processes, compositions, and kits for making bone graft materials for use in bone defect sites are disclosed herein. Also disclosed herein are methods, processes, compositions, and kits for enhancing mammalian bone marrow cells. In some cases, bone loss refers to bone damage such as fractures that require implantable material to stimulate and induce restorative growth of natural bone.

In some embodiments, the method is a method of making a bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome WNT polypeptide; (b). ) The sample is washed to remove the free liposome WNT polypeptide; (c) refers to a method comprising transplanting a bone graft material treated with the ex vivo WNT polypeptide into a bone defect site. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the method is a method of making a bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome WNT3a polypeptide; (b). ) The sample is washed to remove the free liposome WNT3a polypeptide; (c) refers to a method comprising transplanting a bone graft material treated with the ex vivo WNT3a polypeptide into a bone defect site.

In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide. In some embodiments, the method is a method of making a bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome WNT5a polypeptide; (b). ) The sample is washed to remove the free liposome WNT5a polypeptide; (c) refers to a method comprising transplanting a bone graft material treated with the ex vivo WNT5a polypeptide into a bone defect site.

In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide. In some embodiments, the method is a method of making a bone graft material, in which (a) a sample containing mammalian bone marrow cells is ex vivo contacted with a liposome WNT10b polypeptide; (b). ) The sample is washed to remove the free liposome WNT10b polypeptide; (c) refers to a method comprising transplanting a bone graft material treated with the liposome WNT10b polypeptide into a bone defect site.

In some embodiments, the process comprises contacting the isolated mammalian bone marrow cells ex vivo with the liposome WNT polypeptide and the contact time is between about 30 minutes and about 4 hours. Refers to a mammalian bone marrow composition produced by a process that is. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the process comprises contacting the isolated mammalian bone marrow cells ex vivo with the liposome WNT3a polypeptide, and the contact time is between about 30 minutes and about 4 hours. Refers to a mammalian bone marrow composition produced by a process that is.

In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide. In some embodiments, the process comprises contacting the isolated mammalian bone marrow cells ex vivo with the liposome WNT5a polypeptide, and the contact time is between about 30 minutes and about 4 hours. Refers to a mammalian bone marrow composition produced by a process that is.

In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide. In some embodiments, the process comprises contacting the isolated mammalian bone marrow cells ex vivo with the liposome WNT10b polypeptide, and the contact time is between about 30 minutes and about 4 hours. Refers to a mammalian bone marrow composition produced by a process that is.

In some embodiments, the composition is a composition of isolated enhanced mammalian bone marrow cells, which, after treatment with liposome WNT polypeptide, by cells, Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase. , Type I collagen, Axin2, Lip1, and Tcf4, which refers to a composition in which the expression level of one or more of the biomarkers selected from the group is enhanced. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the composition is a composition of isolated enhanced mammalian bone marrow cells, which is treated with liposome WNT3a polypeptide and then by cells, Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase. , Type I collagen, Axin2, Lip1, and Tcf4, which refers to a composition in which the expression level of one or more of the biomarkers selected from the group is enhanced. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide. In some embodiments, the composition is a composition of isolated enhanced mammalian bone marrow cells, which is treated with liposome WNT5a polypeptide and then by cells, Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase. , Type I collagen, Axin2, Lip1, and Tcf4, which refers to a composition in which the expression level of one or more of the biomarkers selected from the group is enhanced. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide. In some embodiments, the composition is a composition of isolated enhanced mammalian bone marrow cells, which is treated with liposome WNT10b polypeptide and then by cells, Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase. , Type I collagen, Axin2, Lip1, and Tcf4, which refers to a composition in which the expression level of one or more of the biomarkers selected from the group is enhanced. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, the composition is also a composition of mammalian bone marrow cells obtained from a 35 year old or older human subject, after treatment with a liposome WNT polypeptide, mammalian bone marrow. A composition in which cells express one or more of biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. Also refers to things. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin. In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide, a Wnt5a polypeptide, or a Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is a Wnt3a polypeptide. In some embodiments, the composition is also a composition of mammalian bone marrow cells obtained from a 35 year old or older human subject, after treatment with liposome Wnt3a polypeptide, mammalian bone marrow. A composition in which cells express one or more of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. Also refers to things. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, the Wnt polypeptide is a Wnt5a polypeptide. In some embodiments, the composition is also a composition of mammalian bone marrow cells obtained from a 35 year old or older human subject, after treatment with liposome Wnt5a polypeptide, mammalian bone marrow. A composition in which cells express one or more of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. Also refers to things. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, the Wnt polypeptide is a Wnt10b polypeptide. In some embodiments, the composition is also a composition of mammalian bone marrow cells obtained from a 35 year old or older human subject, after treatment with the liposome Wnt10b polypeptide, mammalian bone marrow. A composition in which cells express one or more of the biomarkers selected from the group consisting of Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Ref1, and Tcf4 at enhanced expression levels. Also refers to things. In some embodiments, the biomarker is selected from osteocalcin, osteopontin, Axin2, Left1, and Tcf4. In some embodiments, the biomarker is selected from osteocalcin and osteopontin.

In some embodiments, enhanced expression levels are compared to untreated mammalian bone marrow cells. In some cases, enhanced expression levels are about 1% to about 100%, about 1% to about 50%, about 2% to about 20%, about 3% to about 15 compared to untreated mammalian bone marrow cells. %, Or between about 4% and about 10%. In some cases, enhanced expression levels are at least about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8 compared to untreated mammalian bone marrow cells. 5.5%, 9%, 9.5%, or more. In some cases, enhanced expression levels are at most about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, compared to untreated mammalian bone marrow cells. 8.5%, 9%, 9.5%, or less.

In some embodiments, after treatment with the liposome WNT polypeptide, the expression level of the biomarker selected from SOX9 and PPARγ by mammalian bone marrow cells is further reduced. In some cases, reduced levels of expression within mammalian bone marrow cells are compared to untreated mammalian bone marrow cells. In some cases, reduced expression levels are about 1% to about 100%, about 2% to about 80%, about 3% to about 50%, about 3% to about 30 compared to untreated mammalian bone marrow cells. %, Or between about 4% and about 20%.

In some embodiments, the bone marrow cells further comprise a reduced level of apoptosis as compared to untreated mammalian bone marrow cells. In some cases, reduced levels of apoptosis are at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 compared to untreated mammalian bone marrow cells. %, 90%, 100%, 200%, or more. In some cases, reduced levels of apoptosis are at most about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, compared to untreated mammalian bone marrow cells. 80%, 90%, 100%, 200%, or less.

In some embodiments, mammalian bone marrow cells include enhanced bone formation potential as compared to untreated mammalian bone marrow cells. In some embodiments, the enhancement of bone formation potential comprises a new level of bone growth at the site of bone defect after transplantation of treated mammalian bone marrow cells. In some cases, the new bone growth levels of treated and transplanted mammalian bone marrow cells are about 1% to about 20% or about 20% or about 20% compared to the new bone growth levels of transplanted untreated mammalian bone marrow cells. It is between 5% and about 12%. In some cases, the new bone growth levels of treated and transplanted mammalian bone marrow cells are approximately 1.5-fold and approximately 2-fold compared to the new bone growth levels of transplanted untreated mammalian bone marrow cells. , About 3 times, about 4 times, about 5 times, about 10 times, about 20 times, about 50 times, about 100 times, or more.

As used herein, bone graft material refers to a cell composition obtained from a donor, which may be a living body or a corpse. Bone transplant material typically comprises a complex cell population, including stem cells such as mesenchymal stem cells, and optionally also bone cells and their progenitor cells. In some cases, the cells are attached or non-attached bone marrow cells. In some cases, the bone marrow cells are adherent bone marrow cells. In some cases, attached bone marrow cells are bone marrow stem cells or bone marrow progenitor cells. In some cases, adherent bone marrow cells are bone marrow stromal cells. In some embodiments, the donor is an allogeneic donor. In some cases, the donor is a self-donor in the light of the recipient. The amount of cells for bone grafting can vary depending on the donor, recipient, purpose of transplantation, and the like. Bone graft, up to about ^103, up to about ^104, up to about ^105, up to about ^106, up to about ^107, up to about ^108, more than a maximum of about ^109, up to about ^{10 to 10} or containing cells Can include.

Bone implant material is obtained from a donor, such as the iliac crest, anterior ramus (jaw region), and the femur and / or tibia, fibula, ribs, anterior ramus; the part of the spine, eg, during surgery. Obtained from reaming, aspiration, and perfusion of excised parts, bones of the body, etc. In some cases, the graft material is taken from the bone marrow, eg, bone marrow scraped from the endosteal surface of the appropriate bone, or from a block graft containing small blocks of bone marrow and bone. In some cases, allografts are taken from the corpse, bone bank, etc., for example, from the femoral head by hip replacement. In some cases, the bone graft material is freshly harvested or stored at low temperature until needed, as is known in the art.

In some embodiments, bone graft cells are suspended in a suitable culture medium in the presence of an effective dose of liposome Wnt polypeptide, such as L-Wnt3a, L-Wnt5a, or L-Wnt10b. Any suitable medium such as DMEM, RPMI, PBS can be used. Cells, the concentration to maintain viability in the incubation procedure, for example, re about 10 up to ^four per 1ml, about 10 ⁵ maximum per 1ml, about ¹⁰⁶ maximum per 1ml, up to about 10 ⁷ per 1ml Suspension is typical.

In some cases, the contact temperature is between about 0 ° C. and about 37 ° C., or between about 20 ° C. and about 25 ° C. In some embodiments, the contact temperature is low, for example, up to about 32 ° C, up to about 25 ° C, up to about 15 ° C, up to about 10 ° C, up to about 8 ° C, up to about 4 ° C, up to about 1 ° C. However, it is typically above freezing, unless specifically prepared for cold storage. In some cases, the incubation temperature is at least about 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, or higher. In some cases, the incubation temperature is at most about 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, or less.

In some cases, the bone graft material is with the Wnt polypeptide for at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, and up to about 36 hours, up to about 24 hours. Contact for up to about 18 hours, up to about 15 hours, up to about 12 hours, up to about 8 hours, up to about 6 hours, or up to about 4 hours.

The effective dose of Wnt polypeptide may vary depending on the source, purity, preparation method and the like. In some cases, the effective dose of Wnt polypeptide is at least about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0. 9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4.5 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or more. In some cases, the effective dose of Wnt polypeptide is at most about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml. ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4. 5 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or less.

In some embodiments, the Wnt polypeptide is L-Wnt3a and the effective doses of Wnt3a steps are at least about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg /. ml, 0.2 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3. 5 μg / ml, 4.0 μg / ml, 4.5 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or it. It's super. In some cases, the effective dose of Wnt3a polypeptide is at most about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml. , 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0 .9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4.5 μg / Ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or less.

In some cases, the Wnt polypeptide is L-Wnt5a and the effective doses of the Wnt5a polypeptide are at least about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0. .2 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / Ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml 4.0 μg / ml, 4.5 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or more. .. In some cases, the effective dose of Wnt5a polypeptide is at most about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml. , 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0 .9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4.5 μg / Ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or less.

In some cases, the Wnt polypeptide is L-Wnt10b and the effective doses of the Wnt10b polypeptide are at least about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0. .2 μg / ml, 0.25 μg / ml, 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / Ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml 4.0 μg / ml, 4.5 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or more. .. In some cases, the effective dose of Wnt10b polypeptide is at most about 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.15 μg / ml, 0.2 μg / ml, 0.25 μg / ml. , 0.3 μg / ml, 0.35 μg / ml, 0.4 μg / ml, 0.45 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0 .9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 3.0 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4.5 μg / Ml, 5.0 μg / ml, 7.5 μg / ml, 10 μg / ml, 15 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, or less.

In some embodiments, the bone graft material is incubated with the Wnt polypeptide for a period sufficient to enhance bone formation potential. Enhancement can be measured in a variety of ways, including the use of the biomarkers described herein, for example by increased expression of Axin2, and the fibrotic activity within the bone implant material. It can be measured by increasing (measured about 2 to about 6 days after transplantation), by increasing bone formation after transplantation, or by increasing expression of Runx2 or osteocalcin. It can also be measured by reducing apoptosis after transplantation, or by the volume of bone produced after transplantation.

After incubation, the bone graft material can be transplanted to the recipient, for example, after conventional protocols for spinal repair, fractures, dental supports, and the like.

In some embodiments, the Wnt-treated bone graft material is subjected to a lavage step to remove any free liposomal Wnt polypeptide prior to transplantation back into the patient. In some embodiments, at least about 1, 2, 3, 4, 5 or more washes are performed. In some embodiments, at most about 1, 2, 3, 4, 5 or less washes are performed. In some embodiments, any suitable buffer, such as phosphate buffered saline, is used for cleaning.

In some cases, the liposome Wnt remaining after the wash step is at most about 0.01 pg (picogram), 0.05 pg, 0.1 pg, 0.5 pg, 1 pg, 1.5 pg, 2 pg, 2.5 pg, 3 pg, 3.5 pg, 4 pg, 4.5 pg, 5 pg, 5.5 pg, 6 pg, or less. In some cases, the liposome Wnt remaining after the wash step is at least about 0.01 pg (picogram), 0.05 pg, 0.1 pg, 0.5 pg, 1 pg, 1.5 pg, 2 pg, 2.5 pg, 3 pg, 3 pg, 3 pg. .5 pg, 4 pg, 4.5 pg, 5 pg, 5.5 pg, 6 pg, or more.

In some cases, the methods described herein for treating bone graft material with Wnt are for the number of exposures of the Wnt polypeptide to cells, the duration of exposure, the concentration of exposure, and / or toxicity. Allows control. In some cases, the methods described herein for treating bone graft material with Wnt3a are for the number of exposures of the Wnt3a polypeptide to cells, the duration of exposure, the concentration of exposure, and / or toxicity. Allows control.

In some embodiments, restoration of bone formation potential into aged bone grafts is accomplished by incubation with a Wnt polypeptide such as, for example, L-Wnt3a, L-Wnt5a, or L-Wnt10b. In some cases, liposomal Wnt3a treatment reduces cell death in aged bone grafts. In some cases, after transplantation, bone grafts treated with liposome Wnt3a produced more bone (p <0.05). In some cases, liposomal Wnt3a treatment enhanced cell survival within the graft and reestablished the ability of the graft to form bone from aged animals.

In some embodiments, mammalian bone marrow cells are obtained from any mammal, including but not limited to humans. In some cases, the donor is a rodent such as a mouse or rat. In some cases, the donor is a rabbit. In some cases, the donor is human. In some cases, the donor is the recipient himself and the bone marrow cells are autologous bone marrow cells. In some cases, autologous bone marrow cells include attached and non-attached bone marrow cells, as described elsewhere herein. In some cases, autologous bone marrow cells are adherent bone marrow cells. In some cases, autologous bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some cases, autologous bone marrow cells include bone marrow stromal cells. In some cases, the donor is another individual and the bone marrow cells are allogeneic bone marrow cells. In some cases, allogeneic bone marrow cells are obtained from living or cadaveric donors. In some cases, allogeneic bone marrow cells include attached and non-attached bone marrow cells, as described elsewhere herein. In some cases, allogeneic bone marrow cells are adherent bone marrow cells. In some cases, allogeneic bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. In some cases, allogeneic bone marrow cells include bone marrow stromal cells. In some cases, the donor is an individual 35 years or older. In some cases, bone marrow cells obtained from individuals 35 years or older are considered to be senile bone marrow cells or senile bone marrow cells. In some embodiments, the donor is an individual younger than 35 years of age. In some cases, bone marrow cells obtained from individuals younger than 35 years are considered to be juvenile bone marrow cells. In some cases, bone marrow cells obtained from individuals 35 years or older are autologous bone marrow cells or allogeneic bone marrow cells. In some cases, bone marrow cells obtained from individuals younger than 35 years are autologous bone marrow cells or allogeneic bone marrow cells. As used herein, the terms "individual," "subject," "donor," and "patient" as used herein are used interchangeably and include, but are not limited to, any human. Means mammals.

In some embodiments, the methods, compositions, processes, and kits described herein are bone grafts derived from older patients, and other patients with reduced curability, such as smoking. It provides a clinically applicable, regenerative medicine-based strategy for reactivating bone grafts from patients such as those with illness, diabetes, or malnutrition.

Methods for determining the expression level or presence of biomarkers such as Runx2, Osterix, osteocalcin, osteopontin, alkaline phosphatase, type I collagen, Axin2, Left1, Tcf4, SOX9 and PPARγ are well known in the art. In some cases, the expression level or presence of biomarkers can be determined, for example, by flow cytometry, immunohistochemistry, Western blot, immunoprecipitation, magnetic bead selection, and quantification for cells expressing any of these biomarkers. Measured by. Biomarker RNA or DNA expression levels could be measured by RT-PCR, Qt-PCR, microarrays, Northern blots, or other similar techniques. The terms "biomarker" and "marker" as used herein are used interchangeably.

As disclosed herein, determination of the expression or presence of a biomarker of interest at the polypeptide or nucleotide level is accomplished using any detection method known to those of skill in the art. It is intended to determine the expression level or presence of a biomarker protein or gene in a biological sample by "detection of expression" or "detection of levels of". Thus, "detection of expression" determines that a biomarker is not expressed, is not expressed to a detectable extent, is expressed at a low level, is expressed at a normal level, or is overexpressed. Inclusive of cases.

In certain embodiments, the expression or presence of these diverse biomarkers and any clinically useful prognostic markers in biological samples is expressed, for example, by immunohistochemistry, or in situ hybridization and RT. -Detect at the protein or nucleic acid level using nucleic acid-based techniques such as PCR. In some embodiments, the determination of expression or presence of one or more biomarkers is a means for nucleic acid amplification, means for nucleic acid sequencing, means utilizing nucleic acid microarrays (DNA and RNA), or. It is performed by means for in situ hybridization using a specifically labeled probe.

In other embodiments, determination of the expression or presence of one or more biomarkers is performed by gel electrophoresis. In one embodiment, the determination is performed by transcription to a membrane and hybridization with a specific probe.

In other embodiments, determination of the expression or presence of one or more biomarkers is performed by diagnostic imaging methods.

In yet another embodiment, determination of expression or presence of one or more biomarkers is performed with a detectable solid substrate. In one embodiment, the detectable solid substrate is paramagnetic nanoparticles functionalized by an antibody.

In some embodiments, biomarker expression is detected at the protein level, for example, using an antibody that directs a specific biomarker protein. These antibodies are used in a variety of methods, including Western blotting, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemistry. In some embodiments, biomarker detection is achieved by ELISA. In some embodiments, biomarker detection is achieved by electrochemical luminescence (ECL).

In some embodiments, a binding protein capable of specifically interacting with the expression level of the biomarker protein of interest in a biological sample with the biomarker protein or its functionally active variant. Detect via. In some embodiments, a labeled antibody, its binding portion, or other binding partner is used. As used herein, the term "labeled" is a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. Refers to a compound or composition. In some embodiments, the label is detectable by itself (eg, a radioisotope label or fluorescent label) or, in the case of an enzymatic label, a chemical of a detectable substrate compound or substrate composition. Catalyze change.

Antibodies for detecting biomarker proteins are derived from monoclonal or polyclonal antibodies, or are made synthetically or recombinantly. The amount of complexed protein, eg, the amount of binding protein, eg, the amount of biomarker protein associated with an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methods known to those of skill in the art. decide. Detailed reviews of immunological assay designs, theories, and protocols can be found in numerous textbooks in the art (eg, Ausubel et al. (1995), Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience). , NY); Colligan et al. (1994), Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY)).

The choice of markers used to label the antibody will vary depending on the application. However, those skilled in the art can easily determine the choice of marker. These labeled antibodies are used in immunoassays as well as histological applications to detect the presence of any biomarker or protein of interest. The labeled antibody is a polyclonal antibody or a monoclonal antibody. In addition, antibodies for use in the detection of proteins of interest are labeled with radioactive atoms, enzymes, dye or fluorescent moieties, or colorimetric tags, as described elsewhere herein. Also, the choice of tagging label will depend on the desired detection limit. The enzyme assay (ELISA) typically allows the detection of colored products formed by the interaction of enzyme-tagged complexes with enzyme substrates. Radionuclides used as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Includes Pd-109. Examples of enzymes used as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Dye moieties include, but are not limited to, fluorescein and rhodamine. Antibodies are conjugated to these labels by methods known in the art. For example, the enzyme and dye molecule are conjugated to the antibody via a coupling agent such as dialdehyde, carbodiimide, dimaleimide. Alternatively, conjugation also occurs via the ligand-receptor pair. Examples of suitable ligand-receptor pairs are biotin-avidin or biotin-streptavidin, and antibody-antigen.

As used herein, the term "antibody" is used in the broadest sense and is a fully assembled antibody, antibody fragment capable of binding to an antigen (eg, Fab, F (ab') ₂ , Fv, simply. Chain antibodies, diabodies, chimeric antibodies, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides, including those mentioned above, are targeted.

As used herein, the terms "monoclonal antibody" and "mAb" refer to a substantially uniform antibody population, i.e., a naturally occurring mutation in which the individual antibodies that make up the population can be present in small amounts. Refers to antibodies obtained from the same population, except for.

"Natural antibodies" and "natural immunoglobulins" are typically about 150,000 daltons of heterotetrameric glycoproteins with two identical light (L) chains and two identical weights ("natural antibodies" and "natural immunoglobulins". H) A glycoprotein consisting of chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy chain and each light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain ( _VH ) followed by multiple constant domains at one end. Each light chain has a variable domain ( _VL ) at one end and a constant domain at the other end, the constant domain of the light chain with the first constant domain of the heavy chain. Aligned, the light chain variable domain aligns with the heavy chain variable domain. It is believed that certain amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain.

The term "variable" refers to the fact that the sequences of certain parts of a variable domain differ significantly between antibodies. The variable region imparts antigen binding specificity. However, variability is not evenly distributed throughout the variable domain of the antibody. The variability is aggregated in both the light chain variable domain and the heavy chain variable domain within three segments called complementarity determining regions (CDRs) or hypervariable regions. The highly conservative portion of the variable domain is distributed within the framework (FR) domain. The variable domains of the natural heavy and light chains are each four FR regions, most of which are three CDRs, connecting β-pleated sheet-type structures and optionally forming parts of the loop. Includes FR regions that form a CDR-connected, β-pleated sheet-type configuration. The CDRs within each strand are also tightly retained by the FR region and, together with the CDRs derived from the other strands, contribute to the formation of antigen-binding sites of the antibody (Kabat et al. (1991), NIH Publication No. 1). 91-32
See No. 42, Volume I, pp. 647-669). The constant domain is not directly involved in antibody binding to the antigen, but Fc receptor (FcR) binding, antibody participation in antibody-dependent cellular cytotoxicity (participation), and complement-dependent cellular cytotoxicity. It exhibits various effector functions such as induction of action and degranulation of mast cells.

As used herein, the term "hypervariable region" refers to an amino acid residue of an antibody that contributes to antigen binding. Hypervariable regions are amino acid residues derived from "complementarity determining regions" or "CDRs" (ie, residues 24-34 (L1), 50-56 (L2), and 89-97 within the light chain variable domain. (L3); and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in heavy chain variable domains; Kabat et al. (1991).
, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institute of Health, Bethesda, Md.); And / or amino acid residues derived from "hypervariable loops" (ie, residues within the light chain variable domain). 26-32 (L1), 50-52 (L2), and 91-96 (L3); and (H1), 53-55 (H2), and 96-101 (H3) within the heavy chain variable domain; Clothia and Lesk (1987)
, J. Mol. Biol., Vol. 196: pp. 901-917). "Framework" or "
A "FR" residue is, as it is considered herein, a variable domain residue other than a hypervariable region residue.

An "antibody fragment" comprises a portion of a complete antibody, preferably an antigen-binding or variable region of a complete antibody. Examples of antibody fragments are Fab fragment, Fab fragment, F (ab') 2 fragment, and Fv fragment; Diabody; Linear antibody (Zapata et al. (1995), Protein Eng., Vol. 10, pp. 1057-1062). Includes single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody is two identical antigen-binding fragments, called "Fab" fragments, each of which has a single antigen-binding site and its ability to crystallize easily by its name. Produce an antigen-binding fragment with the remaining "Fc" fragment to reflect. Pepsin treatment yields an F (ab') 2 fragment that has two combining sites and is also capable of cross-linking the antigen.

"Fv" is the smallest antibody fragment containing a complete antigen recognition site and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain under close non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the _VH - _{VL dimer.} In addition, the six CDRs confer antigen binding specificity on the antibody. However, a single variable domain (or half of an Fv containing only three antigen-specific CDRs) still has the ability to recognize and bind to an antigen, albeit with a smaller affinity than the entire binding site. ..

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain ( _CH1). The Fab fragment differs from the Fab'fragment by the addition of several residues at the carboxy terminus of the _CH1 domain of a heavy chain containing one or more cysteines derived from the hinge region of the antibody. As used herein, Fab'-SH is a designation for Fab'where the cysteine residue in the constant domain carries a free thiol group. The Fab'fragment is made by reducing the heavy chain disulfide bridges of the F (ab') 2 fragment. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species is of two distinctly different species called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Can be assigned to one of.

Immunoglobulins can be assigned to different classes, depending on the amino acid sequence of their heavy chain constant domains. There are five major classes of human immunoglobulin: IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Can be further divided into. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 isotypes and human IgG3 isotypes have ADCC (Antibody-dependent Cell-mediated Cytotoxicity) activity.

In some embodiments, the expression or presence of one or more biomarkers or other proteins of interest in a biological sample, eg, a tissue sample, is competing with a radioimmunoassay or enzyme-linked immunosorbent assay (ELISA). Binding enzyme immunoassay assay, dot blot (eg, PromegaProtocols and Applications Guide, Promega Corporation (19)
(1991), Western blot (see, eg, Sambrook et al. (1989), Molecular Cloning, A Laboratory Manual, Volume 3, Chapter 18 (Cold Spring Harbor Laboratory Press, Plainview, NY)), High Performance Determined by chromatography such as liquid chromatography (HPLC) or other assays known in the art. Thus, detection assays involve steps such as, but not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation.

In some embodiments, the expression or presence of one or more of the biomarkers described herein is also determined at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art, including, for example, determining the level of biomarker mRNA in a biological sample. Many expression detection methods use isolated RNA. Any RNA isolation method, not selected in the light of mRNA isolation, is utilized for RNA purification (eg, Ausubel et al. (1987-1999), Current Protocols in Molecular Biology (John Wiley & Sons). , New York)). In addition, processing of large numbers of tissue samples was also performed using techniques well known to those of skill in the art, such as the single-step RNA isolation process disclosed in US Pat. No. 4,843,155. Made easy.

Therefore, in some embodiments, detection of the biomarker or other protein of interest is assayed at the nucleic acid level using a nucleic acid probe. The term "nucleic acid probe" refers to a specifically intended target nucleic acid molecule, eg, any molecule capable of selectively binding to a nucleotide transcript. The probe is synthesized by one of ordinary skill in the art or is derived from a suitable biological preparation. The probe is specially designed to be labeled with, for example, a radiolabel, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or another label or tag discussed above or known in the art. .. Examples of molecules used as probes include, but are not limited to, RNA and DNA.

For example, isolated mRNA is used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays. One method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA encoded by the gene being detected. A hybridization probe is, for example, a full-length cDNA, or portion thereof, at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length and is the biomarker described above herein. , A portion such as an oligonucleotide long enough to specifically hybridize with an mRNA or genomic DNA encoding a biomarker under strict conditions. Hybridization of mRNA with a probe indicates that the biomarker of interest or other target protein is expressed.

In some embodiments, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. .. In an alternative embodiment, for example, within a gene chip array, the probe is immobilized on a solid surface and the mRNA is contacted with the probe. One of ordinary skill in the art will readily adapt known mRNA detection methods for use in the detection of levels of mRNA encoding a biomarker of interest or other protein.

Alternative methods for determining the level of mRNA of interest in a sample are, for example, RT-PCR (see, eg, US Pat. No. 4,683,202), ligase chain reaction (Barany (1991)). Year), Proc. Natl. Acad. Sci. USA, Vol. 88: 189-193), Self-sustained sequence replication (Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA, Vol. 87: 1874-1878). Page), Transcription amplification system (Kwoh et al. (1989), Proc. Natl. Acad. Sci.
USA, Vol. 86: pp. 1173-1177), Q-Beta Replicase (Lizardi et al. (198)
8 years), Bio / Technology, Vol. 6: p. 1197), rolling circle replication (US Pat. No. 5,854,033), or known to those skilled in the art after a nucleic acid amplification process by any other nucleic acid amplification method. Accompanied by the detection of amplified molecules using the technique of. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very small numbers. In certain aspects of the invention, biomarker expression is assessed by quantitative fluorescent RT-PCR (ie, TaqMan0 System).

The expression level of the RNA of interest can be a membrane blot (such as a membrane blot used in hybridization analysis such as Northern analysis, dot analysis), or microwells, test tubes, gels, beads, or fibers (or bound nucleic acids). Monitor using any solid support, including. U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, which are incorporated herein by reference in their entirety. And see Nos. 5,445,934. Detection of expression also includes the use of a nucleic acid probe in solution.

In some embodiments, microarrays are used to determine the expression or presence of one or more biomarkers. Microarrays are particularly well suited for this purpose because of their reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of expression levels of multiple genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized with complementary probes on the array and then detected by laser scanning. The intensity of hybridization for each probe on the array is determined and converted to quantitative values representing relative gene expression levels. U.S. Pat. Nos. 6,040,138, 5,800,992, and 6,020,135, 6,033,860, incorporated herein by reference in their entirety. , And Nos. 6,344,316. High density oligonucleotide arrays are particularly useful in determining gene expression profiles for large numbers of RNAs in a sample.

Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in US Pat. No. 5,384,261, which is incorporated herein by reference in their entirety. In some embodiments, the array is made on a surface of virtually any shape, yet on a number of surfaces. In some embodiments, the array is a planar array surface. In some embodiments, the array is a peptide or nucleic acid on beads, on a gel, on a polymer surface, on a fiber such as an optical fiber, on glass, or on any other suitable substrate (each of which is whole of them). 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5, US Pat. 800, 992) is included. In some embodiments, the array is packaged in a manner that allows diagnostic methods or other manipulation of the device, including all.

D. Kits / Products In certain embodiments herein, kits and products for use with one or more of the methods, processes, and compositions described herein are disclosed. Such kits are one or more containers, such as vials, test tubes, etc., each containing one of the individual elements used in the methods described herein. Includes carrying cases, packages, or containers partitioned to receive. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the container is made of a variety of materials, such as glass or plastic.

The products presented herein contain packaging materials. Examples of packaging materials include, but are not limited to, bottles, test tubes, bags, containers, bottles, as well as formulations of choice and any packaging material suitable for the intended dosing regimen and treatment.

For example, the container contains an L-Wnt polypeptide. Such kits optionally include instructions for identification or labeling, or instructions for their use in the methods described herein.

The kit typically includes a label enumerating the contents and / or instructions for use, as well as a package insert with instructions for use. It also typically includes a set of instructions.

In one embodiment, the label is affixed on or attached to the container. In one embodiment, if the letters, numbers, or other symbols forming the label are glued, cast, or etched on the container itself, the label is affixed onto the container; the label. Also accompanies the container, if present as an attachment, for example, in a receptacle or carrying case that holds the container. In one embodiment, the label is used to indicate that the content is to be used for a particular therapeutic application. The indication also points to instructions for use of the contents, such as in the methods described herein.

Further details of the present invention are presented in the following non-limiting examples.

(Example 1)
Production and Purification of Human Wnt3a Polypeptide Human WNT3A is a lipid-modified human stem cell growth factor that is effective in activating adult stem cells and stimulating their self-renewal and survival. be. The 352 amino acid polypeptide of native human WNT3A has been post-translated modified by glycosylation and palmitoylation. Hydrophobic polypeptides can be difficult to purify to homogeneity at scalable levels.

As described herein and described in FIG. 1, human WNT3A is secreted from CHO cells and purified using an ion exchange (Blue Sepharose) column. After purification, recombinant human WNT3A is reconstituted into lipid vesicles consisting of DMPC and cholesterol. Sucrose density gradient data support the physical association between WNT3A and liposomes. In addition, the liposomal human Wnt3a polypeptide (L-WNT3A) can be stable under physiological aqueous conditions. In some cases, the formation of liposome structures is not essential for this interaction. In some cases, lipids with random structural features can interact with human WNT3A to stabilize the polypeptide in an aqueous environment.

In some embodiments, the L-WNT3A formulation is to be used in an investigational new drug (IND) Phase I study to treat bone defects in patients at high risk of delayed bone healing. Optionally, autologous bone graft material (BGM) is harvested, treated ex vivo with L-WNT3A, then washed and pelleted. In some cases, the resulting material, activated BGM (eg, BGM ^ACT ), is considered a medicinal product and is ready for immediate use. In some cases, L-WNT3A will not be administered directly to the patient and will be used to activate autologous cells ex vivo. In some cases, early in the program, the formulation is expected to meet the US Food and Drug Administration (FDA) criteria (purity, stability, etc.) approved for systemically administered liposome protein formulations.

Production of WNT3A from CHO cells Human WNT3A polypeptide was introduced into the Chinese hamster ovary cell line and grown as adherent cells at temperatures ranging from 32 to 37 ° C. To assess whether CHO cells in suspension secrete WNT3A efficiently, CHO cells grown in suspension were examined (FIG. 9).

CHO cells transformed with WNT3A were grown in DMEM supplemented with Glutamax, antibiotics, and serum. The WNT3A plasmid was used as an inducible plasmid, and the concentration of doxycycline in a certain range was examined (Fig. 10). It was observed that doxycycline concentration could modulate the amount of Wnt3a secreted by CHO cells. The cells could grow for up to 10 days, after which a new aliquot of cells was used.

Fetal bovine serum (FBS) was used for the secretion of WNT3A from CHO cells (FIG. 11).

Acclimated medium (CM) from CHO cell cultures was collected, pooled and stored at 4 ° C. for up to 2 months without loss of activity (FIG. 12).

Prior to purification, CM was filtered (0.22 micron filter) and 1% Triton X-100 was added. CM (40 mL) was applied to a Blue Sepharose column (1.6 ml) equilibrated in 20 mM TrisCl pH 7.5 and 1% CHAPS. Flow-throughs were collected and the columns were washed with 4 column volumes of 20 mM TrisCl pH 7.5, 1% CHAPS, 50 mM KCl. The column was washed again with 4 column volumes of 20 mM TrisCl pH 7.5, 1% CHAPS, 100 mM KCl. The column was then washed with 3 column volumes of 20 mM TrisCl pH 7.5, 1% CHAPS, 150 mM KCl, where human WNT3A was eluted from this wash. The column was washed again with 4 column volumes of 20 mM TrisCl pH 7.5, 1% CHAPS, 250 mM KCl. The yield from 40 mL of CM was 12 μg of a polypeptide with a purity of approximately 60%. Compared to other purification schemes, this method resulted in WNT3A in high purity and high yield. In some cases, the purity was 70%.

WNT3A production and liposomes WNT3A characterization: SDS-PAGE / Western blot: For SDS PAGE analysis and staining analysis with Coomassie blue, purified fractions of WNT3A were collected and stained with Coomassie Brilliant Blue and silver nitrate. , 12% SDS polyacrylamide gel was used to analyze the purity of the WNT3A polypeptide (FIG. 2). As shown in FIG. 2, purified fractions were collected and analyzed for the purity of WNT3A polypeptide by 12% SDS polyacrylamide gel stained with Coomassie Brilliant Blue and silver nitrate. The estimated purity of WNT3A was 70%. Total protein concentration was measured using the Pierce 660 protein assay kit and absorbance at 280 nm. For each assay, a BSA calibration curve was created to estimate total protein concentration. WNT3A polypeptide concentration was determined using quantitative Western blot and WNT3A calibration curve.

Mass Spectrometry: Mass spectrometry analysis verified that the WNT3A polypeptide was lipid-added and glycosylated (FIG. 3). The polypeptide was observed to be palmitoylated in S209.

As shown in FIG. 3, the method for preparing a WNT3A sample for mass spectrometry was as follows. The polypeptide sample was precipitated using 4 times the volume of acetone pre-cooled at −80 ° C. Samples were stored overnight at −80 ° C. and then subjected to ultracentrifugation at 4 ° C. and 10,000 RPM for 10 minutes. The supernatant was removed and the polypeptide was reconstituted in a solution containing 8M urea and a protease mix (Promega). Samples were reduced with 5 mM DTT and incubated at 55 ° C. for 30 minutes. The sample was then cooled to room temperature and then alkylated with acrylamide (15 mM) at room temperature for 30 minutes. The sample was then diluted with 50 mM ammonium bicarbonate, pH 8.0 to a concentration of urea <1 M and 1 μg of trypsin protease (Promega) was added. Digestion was performed overnight at 37 ° C. The reaction was quenched by adding 50% formic acid, the peptides were rapidly cleaned and concentrated on a C18 microcolumn (NEST group).

Liquid Chromatography and Mass Spectrometry (LCMS): Peptides were reconstituted in mobile phase A. Waters NanoAccity LC was used, running at 300 nL / min and using a gradient for lengths between 80-120 minutes. The analysis column was filled and prepared in-house with fused silica (inner diameter 75 um) using a PEEKE C183 3 um matrix with a length of approximately 15 cm. Fragmentation of high intensity precursor ions by MSMS was performed using an LTQ Orbitrap Velos mass spectrometer set to data dependent acquisition (DDA) mode. CAD fragmentation, HCD fragmentation, and ETD fragmentation methods were used.

Database Search and Analysis: Using a Quest, from a mass spectrometer. RAW files were searched against the Human Uniprot database. Next, a custom database containing the WNT3A sequence was created and searched to allow a series of variable modifications, not limited to STY phosphorylation, ST hexNac, STNac, STC palmitoylation, and the like. All files retrieved by the Quest were uploaded to Scaffold (Proteome Software) for scrutiny and semi-quantitative analysis.

For positive controls (WNT3A protein purchased from StemRD), approximately 43% coverage was achieved. Sequence coverage and unique peptides were highlighted in yellow (Fig. 3A). Modified residues are shown in green. The palmitoylation modification was detected only on the C77 residue (Fig. 3B). In the case of purified WNT3A, coverage of about 51% was achieved. Sequence coverage and unique peptides were highlighted in yellow (Fig. 3C). Modified residues are shown in green. Palmitoylation modifications were detected only on residues S211 (FIGS. 3C, D), and possible myristoylation modifications were detected on residues S139 and S181.

L-WNT3A formulation and characterization

Net positive by incubating positively charged, negatively charged, or charge-neutralized lipids with purified Wnt3a at room temperature for a minimum of about 6 hours to a maximum of about 24 hours. Liposomal Wnt3a (L-Wnt3a) polypeptides containing a charge, a negative charge, or a neutral charge were produced. The lipid-to-cholesterol ratio was maintained at 90:10. After incubation, L-Wnt3a was separated from CHAPS and other impurities by centrifugation at 4 ° C. with a force in the range of 16,000 to 150,000 xg for up to 1 hour. L-Wnt3a pellets were then resuspended in sterile 1x PBS, covered with nitrogen gas and stored at 4 ° C.

Lipid Interactions with WNT3A: When combined with lipids of varying carbon chain lengths, WNT3A confirmed a functionally involved interaction with uncharged lipids (FIG. 15). These data suggest that the interaction was primarily a hydrophobic interaction that mediated the association between the WNT3A polypeptide and the liposome (Fig. 4).

Characterization of L-WNT3A by TEM: TEM was used to visualize the range of diameters observed for L-WNT3A (FIG. 4). The appearance and size of L-WNT3A falls within the size range reported for exosomes, eg, 20-100 nM. See also Dhamdhere et al. (2014), PLoS ONE, Vol. 9 (No. 1): e83650, which is incorporated herein by reference in its entirety.

Mouse LSL cell-based assay: To normalize beta-galactosidase activity against cell number, mouse LSL cells were subjected to the Wnt-responsive luciferase reporter plasmid, pSuperTOPFlash (Addgene), and the constitutive LacZ expression construct, pEF /. Myc / His / LacZ (Invitrogen) was stably transfected. The above two plasmids were stably transfected into human fetal-derived renal epithelial (HEK293T) cells. Unless otherwise stated, cells (50,000 cells per well, 96 well plates) were supplemented with 10% FBS (Gibco) and 1% P / S (Cellgro). DMEM was treated with L-WNT3A at a concentration of 10 uL in a total volume of 150 uL. Also included was a dilution series of purified WNT3A polypeptides.

Cells are incubated overnight at 5% CO ₂ , 37 ° C., then washed, lysed with Applied Biosystems and dual-light.
Expression levels of luciferase and β-galactosidase were quantified using a combined reporter gene assay system (Applied Biosystems). Bioluminescence was quantified by triple reading on a dual light ready luminometer (Berthold). The activity of WNT3A (ng / uL) and L-WNT3A was defined from the calibration curve prepared by serial dilution of the WNT3A polypeptide (FIG. 5). In experiments over time, WNT3A activity was expressed as a percentage of activity. The percentage of activity was calculated as follows:

Summary of Stability Data Based on the LSL reporter cell assay and PAGE-Western blot analysis, the fermentation-derived conditioned medium was stable at 4 ° C. for a minimum of 2 months. Based on the LSL reporter cell assay and PAGE-Western blot analysis, L-WNT3A was stable at 4 ° C. for> 106 days (Fig. 6). Based on the LSL reporter cell assay and PAGE-Western blot analysis, at 23 ° C., the half-life of L-WNT3A was> 48 hours (blue line, FIG. 7). Compare with a liposome-free WNT3A polypeptide that lost its activity within 5 minutes at 23 ° C.

Based on the LSL reporter cell assay and PAGE-Western blot analysis, the half-life of L-WNT3A was 10.5 hours at 37 ° C. (blue line, FIG. 8). Note that even with the addition of detergent (CHAPS) to stabilize the hydrophobic polypeptide, WNT3A lost its activity within 5 minutes (red line, FIG. 8).

Characterization of WNT cell reporter assay The LSL reporter cell line was used to assess the activity of Wnt polypeptides and agonists. The following characterization for L-WNT3A was performed using the following reporter assay. Using the LSL reporter cell line and the HEK293 reporter cell line, cells were ^{seeded at 5.0 × 10 4} cells per assay well and the activity of WNT3A and L-WNT3A was quantified. Under these conditions, WNT3A and L-WNT3A showed similar ability to activate Wnt signaling (FIG. 13).

Accuracy and detection limit: The sensitivity of the assay is R ² = 0.99. In FIGS. 14A and 14B, WNT was shown to reach an optimal concentration of 0.1 ng / μL. The assay cutoff points were 0.025 ng / μL and 0.2 ng / μL. The LSL assay was unaffected by intentional fluctuations in WNT3A concentration (Fig. 14C). The range of WNT3A concentrations supported a proportional luciferase response over time, indicating the reliability of the assay.

Specificity was supported using R-spondin 2, a Wnt polypeptide agonist that activates the WNT pathway by synergistic action with WNT polypeptides. The specificity of the assay was also supported by the use of WNT5A, which suppresses activation of beta-catenin-mediated pathways in the presence of the Wnt co-receptor Ror2. As shown in FIG. 13D, none of the reagents was confirmed to be active in the LSL reporter assay.

As shown in FIGS. 13 (A, B), cells were ^{seeded at 5.0 × 10 4} cells per assay well and both WNT3A activity and L-WNT3A activity were quantified. Under these conditions, both WNT3A and L-WNT3A showed similar ability to activate Wnt signaling. The sensitivity of the assay is R ² = 0.99. Purified human WNT3A polypeptide was used as a positive control. (C) The LSL assay was unaffected by intentional fluctuations in WNT3A concentration. Data from a range of concentrations confirmed a proportional luciferase response over time, which provided an indicator of assay reliability. (D) Specificity was supported by the addition of Rspo2 in the presence and absence of WNT3A.

Accuracy: The coefficient of variation within and between assays of the assay was 17.2%.

Dose-Response Curve for L-WNT3A Using Primary MEF LSL and HEK293T cells were engineered to be sensitive to Wnt and Wnt agonists, thereby the relationship between dose, drug effect, and clinical response. Provided modest but meaningful data about. To more closely mimic the cellular response to Wnt stimulation in vivo, expression of Wnt's target gene, Axin2, was assayed for mouse embryonic fibroblasts (MEFs) using as a measure of pathway activity.

Within the primary cells, the linear range of effective concentrations was 0.025-0.1 ng / μL WNT3A (FIG. 14A). We also assayed for L-WNT3A activity in bone marrow-derived stem cells and found that the linear range of effective concentrations ranged from 0.004 to 0.08 ng / μL (FIG. 14B). Therefore, the primary cell population exhibited significantly different sensitivities to Wnt stimuli.

WNT3A can be maintained by substituting CHAPS with more removed lipids. DMPC and cholesterol lipids could replace CHAPS and maintain WNT3A activity. Liposomes made with other lipids, such as MPPC, DPPC, DMPS, DMPG, and DMGE, were inactive (FIG. 15).

Reconstitution of WNT3A with Lipids Many proteins are denatured at high temperatures, and avoiding such denaturation at body temperature is the key to extending the duration of protein therapeutics. Liposomal packaging preserves the biological activity of Wnt3a and the formulation may be effective in multiple bone injury applications. The affinity of Wnt3a for liposomes and the stability of this association were characterized. The kinetics and kinetics of Wnt pathway activation by L-WNT3A were supported.

Affinity between WNT3A and liposomes: Sucrose density gradient data support the physical association between WNT3A polypeptides and lipids (FIG. 16). The sucrose density gradient of FIG. 16 confirmed the physical association between WNT3A and the lipid. The WNT3A polypeptide was localized to a high density fraction (FIG. 16A), which also showed WNT activity (FIG. 16B). Western analysis confirmed that the majority of WNT3A polypeptides were localized to this high density fraction (Fig. 16C). The phospholipid assay confirmed that this high density fraction contained the majority of lipids (orange line, FIG. 16B). Therefore, when compared to WNT3A alone (divided into low density fractions 2-3; FIGS. 16D, E), L-WNT3A shifts to a high density fraction, which is a physical association with WNT3A. Associations, not as aggregates with liposomes, were supported.

Estimated Affinity Between Wnt3a and Liposomes: The binding affinity between Wnt3a / Frizzled was approximately 3.6 nM. The binding affinity between Wnt3a / liposome was about 9 nM. In a competitive assay between L-Wnt3a and Frizzled, Wnt3a was observed in liposome pellets (FIG. 17A). After further incubation, Wnt3a dissociated from the liposome pellet and preferentially associated with Frizzled in the supernatant (FIG. 17B). Therefore, the affinity of Wnt3a for liposome membranes was smaller than that of Wnt3a for Frizzled.

In a second competitive assay between liposomes and LRP6, LRP6 was observed in the supernatant (FIG. 17C). When LRP6 was incubated in the presence of L-Wnt3a, the LRP6 protein was transferred from the supernatant (FIG. 17C) to the pellet of L-Wnt3a (FIG. 17D).

CONCLUSIONS: Based on these pull-down assays, the binding affinity between Wnt3a and the liposome membrane was estimated to be about 6 nM. These assays also confirmed that the reconstituted Wnt3a within the liposome membrane was arranged in a manner capable of interacting with its reporters Frizzled and LRP6.

Reaction rate of WNT3A association with liposomes Based on the LSL reporter cell assay and PAGE-Western blot analysis, Wnt3a rapidly associates with liposomes, and this association contributed to the maintenance of polypeptide activity. Initially, Wnt3a activity was observed in the supernatant. Over the course of about 30 minutes, the Wnt3a polypeptide was transferred from the supernatant to the pellet at ^{a rate of about 3 × 10 -3} nM / sec (FIG. 18A). By 6 hours, 90% of Wnt3a activity was observed in the pellet. Similarly, Western blot analysis showed that 90% of the polypeptides were found in pellets (Fig. 18B). After 24 hours at 23 ° C., the liposomes were separated from the aqueous supernatant by centrifugation. No visible polypeptide precipitation was observed in the pellet. Western blotting confirmed that the majority of Wnt3a was present in liposome pellets (Fig. 18C). FIG. 18D shows the Wnt3a activity in the supernatant and in the pellet at the end of the experiment.

FIG. 19 illustrates the interaction of Wnt5a and Wnt10b with liposomes (FIG. 19).

Scale-up experiment: Cells from the freezer stockpile were seeded on a 15 cm tissue culture plate. After incubation at 37 ° C. and 5% CO ₂ for 3-4 days, cells were grown at 1: 5 on a 2 x 15 cm plate for 4 days. These cells were further grown 1: 5 on a 20 x 15 cm plate. Twenty-four hours after incubation, cells were induced with doxycycline. CM was collected every 24 hours and stored at 4 ° C. The activity of CM was measured to confirm the secretion of WNT3A. 1% Triton X was added to 1 L of CM and filtered through a 0.22 μm filter. The CM was then loaded onto a 150 ml Blue Sepharose column. From this test, 80 μg of WNT3A was eluted in a KCl gradient resulting in a total yield of 40%.
Material and source CHO cells: Invitrogen
Plasmid vector: Promega
Lipids: Avanti Polar Lipids
Blue Sepharose Purification Column:
WNT3A standard material: Stem R & D
LSL cells:
Ultra Centrifuge:
Abbreviation BGM: Bone graft material BGM ^ACT : L-WNT3A activated bone graft material CHO: Chinese hamster ovary CM: conditioned medium Fz: Frizzled, which is a WNT receptor
HEK: Human fetal kidney LSL: Mouse L cell
L-WNT3A: Liposome WNT3A
MEF: Mouse embryonic fibroblasts

(Example 2)
The human Wnt3a polypeptide was produced via transfection of a genetically engineered Chinese hamster ovary (CHO) cell line with a plasmid carrying the human Wnt3a nucleic acid. A lipofectin-based transfection method was used. The plasmid was placed under the control of a doxycycline-inducible promoter. Wnt3a-transformed CHO cells in adherent culture or DMEM (Dalbeco modified Eagle's medium) + 10% fetal bovine serum (FBS), GlutaMax ™ (glutamine-based dipeptide), antibiotics (eg, doxicycline) ), G418, non-essential amino acids, blasticidine, and suspension culture in culture medium containing serum, grown at temperatures ranging from about 32-37 ° C. The amount of Wnt3a secreted by CHO cells depended on whether they were grown as adherent cultures or suspension cultures. Doxycycline was added to the medium at a concentration within the range that could modulate the amount of human Wnt3a produced by CHO cells. The culture medium contained fetal bovine serum (FBS), which was present throughout the culture period during which CHO cells secreted Wnt3a. The cells could grow for up to 10 days, after which a new aliquot of cells was used.

Compared to purification schemes that use continuous gradients or gel filtration, this purification scheme uses a stepwise gradient, which results in high purity and high yields, as shown in Tables 1 and 2. In purification schemes using continuous gradient or gel filtration, a continuous salt gradient was run on a Blue Sepharose column over 150 mM to 1.5 M NaCl and then run on a gel filter and a heparin sulfate column. Optionally, as described elsewhere herein, the scheme also utilizes hydrophobic purification steps such as hydrophobic columns (eg, protein A-Sepharose columns).

Table 1 shows the results obtained by the purification steps of the present invention, and Table 2 shows the results obtained by a purification scheme using a continuous gradient purification step or a gel filtration purification step.

(Example 3)
All animal procedures followed the protocol approved by the Stanford Committee on Animal Research. Mice with wild-type beta-actin-enhanced green fluorescent protein (ACTB-eGFP; The Jackson Laboratory, Sacramento, California) and CD1 were used. Mice <3 months of age were considered young and mice> 10 months of age were considered old. Axin2 ^{CreERT / +} and R26R ^{mTmG / +} mice were purchased from Jackson Labs. Old-age wild-type Lewis rats (Charles Rivers, MA "retired breeders") were used for spinal fusion in accordance with the AAUC and IUPAC guidelines.

Recovery and Treatment of Bone Graft Material (BGM) for Rodent Animal Models In this study, both rats and mice were employed. Although the use of mouse models allows for a wide range of molecular analyzes, autologous grafts are highly invasive to these small animals, so rats should be used when performing autologous transplants ( For example, FIGS. 20 and 25), syngeneic mice were used when adopting advanced molecular techniques (FIGS. 21-24). In all cases, the bone implant material (BGM) is torn in the longitudinal direction of the bone, the endosteal surface is gently scraped with a sharp instrument, and the bone marrow contents are perfused into the recovery dish to allow the femur. , Tibia, and iliac crest. This method mimicked the reamer / perfusion / aspiration (RIA) method used in humans.

Axin2 ^{CreERT / +;} To induce recombination in R26R ^{mTmG / +} mice (Fig. 21), animals were given 160 μg of tamoxifen per gram of body weight via IP injection or gavage over 5 consecutive days. Tissues were harvested 7 days after the first treatment and analyzed by GFP immunostaining or fluorescence.

To ensure that BGM aliquots for subcapsular (SRC) transplantation are comparable in terms of cell content, pool BGM from 3 mice (littermates) and then in the transplantation assay. In exactly the same way as above, it was divided into 20 μL aliquots. The DNA contents were extracted with a DNeasy Tissue Kit (QIAGEN) and the relative DNA concentration was measured using a Quant-iT PicoGreen dsDNA Kit (Invitrogen) and a microplate fluorescence reader (BERTHOLD, Bad Wildbad, Germany). The percent variation in DNA content was <20% (FIG. 26).

In ^{order to obtain BGM ACT} , freshly collected BGM is subjected to liposome formulation with phosphate buffered saline (L-PBS) or liposome formulation with WNT3A (L-WNT3A; effective concentration of L-WNT3A = 0.15 μg / It was placed in 20 μL of culture medium containing mL) and maintained at 23 ° C. for 1 hour.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Tissue samples were homogenized in TRIzol solution. RNA was isolated and reverse transcription was performed. Quantitative real-time PCR was performed using the Prism 7900HT Sequence Detection System and Power SYBR Green PCR Master Mix (Applied Biosystems). The level of gene expression was determined by the ΔΔCT method and standardization in the light of their GAPDH values. All reactions were performed in triplicate and mean and standard deviations were calculated.

The primer sequences (from 5'to 3') are as follows: Axin2, [for-TCATTTTCCGAGAACCACCGC], [rev-GCTCCAGTTTCAGTTTTCCAGCC]; -ACCCAGAAGACTGTGGATGG [rev-GGATGCAGGGATGATGTTCT]; ALP [for-ACCTTGACTGTGGTTACTGC, [rev-CATATAGGATGGCCGTGAAGG]; Osterix, [for-GGAGACCTTGCTCGTAGATTTC], [rev-GGGATCTTAGTGACTGCCTAAC]; Osteocalcin, [for-TGTGACGAGCTATCAAACCAG], [rev-GAGGATCAAGTTCTGGAGAGC].

Western analysis BGM is taken from young (N = 5) and old (N = 5) mice, followed by 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. It was placed in Dulbecco's Modified Eagle's Medium (DMEM) containing _{and incubated in 5% CO 2} at 37 ° C. After 24 hours, the non-adherent cells were removed, the medium replaced and the adherent cells were subcultured until they reached confluence. The medium was changed every 3 days. In some experiments, cells after the third passage were treated with L-PBS or L-WNT3A (effective concentration = 0.03 μg / mL). In these experiments, cells were harvested after 24 hours and lysed using RIPA buffer. Total proteins were extracted for Western analysis. Panactin was used as an internal control to confirm protein integrity. Antibodies to WNT3A, non-phosphorylated β-catenin, and Axin2 were used. The integrated intensity was analyzed by ImageJ and the results by Western blotting were quantified.

Subcapsular Transplantation In some cases, a subcapsular assay (SRCA) was used to assay for its differentiation potential. After inhaling the anesthetic into a syngeneic host mouse, a skin incision was made on the left flank immediately caudal to the thorax. The abdominal cavity was opened to expose the kidneys. A small incision was made in the renal capsule and the BGM was carefully placed under the capsule using a flexible plastic tube. The kidneys were then returned to the abdominal cavity and the peritoneum and skin were closed with sutures. Buprenorphine (0.05 mg / kg) was used for analgesia.

When background music was ^{collected from Axin2 CreERT2} ; R26 ^mTmG donors, the host was then given tamoxifen by gavage (100 μL at 10 mg / mL) for 5 days starting on day 0. SRC grafts were collected at the time of indication.

Adenovirus-mediated inhibition of Wnt signaling An adenovirus construct of DKK1 and an adenovirus construct of Fc, a negative control, were generated. The adenovirus construct was transfected into 293T cells. After 2 days, cells were harvested, lysed and precipitated by centrifugation. Purified adenovirus was aliquoted and stored at -80 ° C. In vitro inhibition of Wnt was achieved by 2-hour incubation of BGM with Ad-Dkk1 and control Ad-Fc, followed by transplantation of BGM aliquots into the calvaria defect.

Critical size defect in calvaria Mice were anesthetized and a 3 mm incision was made to expose the parietal bone. A microdissection projectile point was used to create an annular full-thickness defect with a diameter of 2 mm, but the dura was not disturbed. BGM aliquots were incubated with Ad-Dkk1 and control Ad-Fc for 2 hours. BGM aliquots were then implanted in the calvaria defect and the skin was closed with sutures. After recovery from surgery, mice were given buprenorphine for analgesia.

Micro-Computed Tomography (Micro-CT) analysis was performed as follows. Mice were anesthetized with 2% isoflurane and scanned using a multimode positron emission tomography-computed tomography data collection system (Inven PET-CT) with a resolution of 40 μm. The data was analyzed with MicroView software. Structure and bone volume were assigned for each sample using the 3D region of interest tool.

Evaluation of the bone volume fraction of the regenerated product (percentage [BV / TV,%] calculated by dividing the total bone volume by the bone volume of the regenerated product) is performed by high-resolution micro-CT (vivaCT40), as well as It was performed using 70 kVp, 55 μA, an integration time of 200 ms, and an isotropic voxel size of 10.5 μm. The region of interest is 2 cm long and extends from 250 μm proximal to the edge of the defect to 250 μm distal beyond the opposite edge of the defect (total diameter 1.5 cm). Bone was separated from soft tissue using a threshold of 275 mg of hydroxyapatite per cm3. Scanning and analysis complied with published guidelines.

Spinal fusion Anesthetized Lewis rats using a cocktail of 70-100 mg / kg ketamine and 5-10 mg / kg xylazine. The lumbar region of the rat was then shaved and disinfected with gauze soaked in Betadine. Prior to incising the skin, rats were SC / IP injected with 0.02 mg / kg of buprenorphine, an analgesic. First, bone graft material (BGM) was collected from the iliac crest. Briefly, the left iliac spine was palpated, a vertical skin incision was made, and the dorsal ridge of the iliac spine was accessed and exposed by a blunt die section. The attached muscles and periosteum were lifted, and 0.3 g of BGM was collected with osteotomy forceps and distributed in small amounts. BGM was then incubated with 100 μL L-PBS or 100 μL [0.15 μg / mL] L-Wnt3a while exposing the transverse processes.

A posterior blunt die section was performed to expose the transverse process, and the reflexed paraspinal muscles were held in place by a retractor. The periosteum was removed from the transverse processes extending from L4 to L5, and the cortex was removed with a bar. BGM was applied on and between the transverse processes of L4 to L5. The paraspinal muscles were closed with resorbable sutures (4-0 Nylon; Ethicon) and the skin was closed with nodular non-absorbable sutures (4-0 Nylon; Ethicon). The surgical site was treated with an antibiotic ointment. 10 mg / kg Baytril was delivered subcutaneously. Buprenorphine (0.02 mg / kg) was administered for 3 days after surgery, with subsequent dosing as needed to control pain.

Sample Preparation, Tissue Processing, Histology Tissues were fixed in 4% paraformaldehyde (PFA) at 4 ° C. overnight. Samples were decalcified in 19% EDTA for 1 day. Specimens were dehydrated with ascending ethanol series and then embedded in paraffin. Eight micron thick strips were cut out and collected on Superfrost-plus slides for histology. Safranin O staining, aniline blue staining, and gomori staining were performed. Tissue sections were photographed using a Leica DM5000B digital imaging system.

ALP Staining, TRAP Staining, and TUNEL Staining Alkaline phosphatase (ALP) activity includes nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indrill phosphate (BCIP), and NTM buffer (100 mM). Detected by incubation in NaCl, 100 mM Tris pH 9.5, 5 mM MgCl). Tartrate-resistant acid phosphatase (TRAP) activity was observed using a leukocyte acid phosphatase staining kit according to the manufacturer's instructions. After development, slides were dehydrated in a dilution series of ethanol, cleaned in Citrisolv and coverslip treated with Permount encapsulant. Sections were permeabilized using 0.1% Triton X-100 and 0.1% sodium citrate for TUNEL staining and incubated with the TUNEL reaction mixture (In Situ Cell Death Detection Kit). Sections were encapsulated with DAPI encapsulant and visualized under epifluorescence microscopy.

For the bromodeoxyuridine (BrdU) assay, mice were given an intraperitoneal injection of BrdU labeling reagent (Invitrogen, CA, USA) and euthanized 4 hours after injection. The detection of BrdU was performed using the BrdU Staining Kit according to the manufacturer's instructions.

Immunohistochemistry Tissue sections were deparaffinized and rehydrated in PBS. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 5 minutes and then washed in PBS. Slides were blocked with 5% goat serum for 1 hour at room temperature. Appropriate primary antibody was added and incubated overnight at 4 ° C. Samples were then incubated with the appropriate biotinylated secondary antibody and avidin / biotinylated enzyme complex and developed with the DAB substrate kit. Antibodies used included GFP antibody, as well as DLK1 antibody, Runx2 antibody, Sox9 antibody, and PPAR-γ antibody.

Micro-CT analysis and quantitative scanning and analysis of graft growth followed published guidelines. Rats were anesthetized with 2% isoflurane and scanned by a micro-computed tomography data collection system (Inveon) with a resolution of 52 μm. To define the graft growth that occurred within each sample, scan data at time points POD2 and POD49 were exported to OsiriX software version 5.8 and registered in the same orientation for segmentation. The new bone formed was compared to the initial BGM volume transplanted. Differences between datasets were determined by utilizing the Mann-Whitney test within the XLStat software version. A p-value <0.05 was considered statistically significant.

Quantitative and statistical analysis GFP staining, BrdU staining, TUNEL staining, DLK1 staining, osteocalcin staining, and aniline blue staining were quantified. Photoshop CS5 was used to determine the number of pixels in the region of interest (ROI) at the site of injury. The magic wand tool was used to determine the area of positive pixels in the ROI. The ratio of positive signal pixels to ROI pixels was expressed as a percentage. At least 5 sections, evenly spaced across the site of injury, were quantified to determine the average value for each sample. Five samples were included in each group (n = 5). Results are presented as mean ± SD. Differences were quantified using Student's t-test as described herein. P ≦ 0.05 was considered significant.

Bone graft material contains multiple stem / progenitor cell populations, and in some cases, the best anatomical site for harvesting autologous grafts includes the disease status of the donor site and the availability of bone reserves. , Depends on a number of factors. When BGM was taken from three anatomical sites using modified reamer perfusion aspiration (RIA), the femur, iliac crest, and tibia yield BGM with significantly different histological appearances. It was noticed. BGM of the femur contained adipocytes in addition to hematopoietic cells, even when collected from young animals (FIG. 20A). BGM of the iliac crest contained mostly trabecular fragments covered with tightly attached cells (Fig. 20B). BGM derived from the tibia contained a large amount of fibrous interstitium and small annuclear cells (Fig. 20C). Quantitative RT-PCR was used to assess the expression of endogenous bone-forming genes and that of the three sources, iliac crest BGM expresses alkaline phosphatase and osteocalcin at significantly higher levels. Found (Fig. 20D).

In some cases, the bone-forming properties of autologous grafts may be attributed to stem / progenitor cell populations and osteoblasts within the bone graft material (BGM). This bone formation property of autologous grafts was investigated by transplanting BGM into a subcapsular (SRC) assay. SRC supplies blood vessels to the transplanted tissue and supports the differentiation of cells into multiple types of tissue, including bone, skin, muscle, teeth, organs, and tumors. BGM was taken from the iliac crest and then transplanted under the renal capsule of the animal (Fig. 20A) and generated there for 7 days.

Incorporation of BrdU confirmed the high degree of mitotic activity of cells within autologous BGM (Fig. 20B). By immunostaining for Runx2 (Fig. 20C), Sox9 (Fig. 20D), and PPARγ (Fig. 20E), a subset of cells within the BGM were genetic markers associated with commitment to bone formation, chondrogenesis, and fat formation. It was confirmed that it was expressed. On day 7, a subpopulation of BGM-derived cells had differentiated into bone (Fig. 20F), cartilage (Fig. 20G), and fat (Fig. 20H). Taken together, these data support that BGM contains stem / progenitor cells that can differentiate into all three lineages.

Wnt signaling within bone graft material decays with age Axin2 ^CreERT2 ; R26 ^mTmG reporter mice are used to induce recombination, thereby intraperiosteum (FIG. 21A) and endosteum (FIG. 21B). GFP ^{+ ve} pre-osteoblasts within were identified. The frequency of GFP ^{+ ve} cells in the endosteum was about 0.1% (Fig. 21C). GFP ^{+ ve} cells were also identified in freshly harvested BGM (Fig. 21D). Therefore, a subset of cells within a heterologous BGM are Wnt responsive.

The Wnt-responsive state of BGM derived from young (<3 months old) mice was compared with the Wnt responsive state of BGM derived from old (> 10 months old) mice. ^{In BGM collected from aged} mice (BGM aged) by quantitative absolute RT-PCR, the expression of Wnt target genes Axin2 and Left1 is expressed in BGM collected from ^{young mice (BGM young).} In comparison, it was confirmed that it was almost half (Fig. 21F). Western analysis confirmed that the expression of Wnt3a, phosphorylated beta-catenin, and Axin2 in ^{BGM aged was all significantly lower than in BGM young} (Fig. 21G). Therefore, the intrinsic Wnt responsive state of BGM deteriorates with age.

Bone differentiation potential also declines with age In humans, bone healing rates decline with age. A similar age-related decrease in BGM bone formation capacity was also found in mice. Freshly harvested BGM ^aged showed significantly lower expression levels of the bone-forming genes alkaline phosphatase, Osterix, and osteocalcin ^{compared to freshly harvested BGM young} (FIG. 22A).

An SRC assay was used to investigate whether reduced expression of the bone-forming gene affects the bone-forming ability of BGM. However, performing autologous transplantation in mice is clearly traumatic. Synonymous donors and hosts were used to mimic autologous transplantation. Since syngeneic animals are so closely related, their tissues are immunologically compatible and transplantation of tissues does not provoke an immune response. The ACTB-eGFP mice and BGM used as donors were easily identifiable within the SRC by their GFP fluorescence (FIGS. 22B, 22C).

Seven days after transplantation, BGM was collected and analyzed for evidence of bone formation. The aniline blue-stained osteoid matrix was ^{apparent in BGM young} (FIG. 22D) but not in BGM ^aged (FIG. 22E; quantified in FIG. 22F). The ^{osteoid matrix in BGM young} was undergoing calcification as shown by ALP staining (Fig. 22G), whereas BGM ^aged did not show ALP staining (quantified in FIG. 22H; I). .. Furthermore, GFP immunostaining confirmed that the number of surviving donor cells found was similar in both ^{BGM young} (Fig. 22J) and BGM ^{aged (Fig. 22K) (quantified in FIG. 22L).} .. Taken together, these data indicate that BGM-induced bone-forming gene expression and bone-forming ability decline with age.

Wnt signaling and osteogenic potential by BGM The endogenous Wnt responsiveness and osteogenic potential of BGM diminishes with age. To investigate whether reduced Wnt signaling contributes to this age-related decline in bone formation potential, we blocked endogenous Wnt signaling within the BGM. Dkk1, an inhibitor of Wnt overexpression, was also used to transiently eliminate Wnt signaling in vivo. Ad-Dkk1 or Ad-Fc (control) was delivered to the medullary cavity of young mice, then 24 hours later BGM ^age was harvested and aliquots were transplanted into critical size (non-healing) skeletal defects.

Control BGM ^young was strongly positive for ALP activity (FIG. 23A), and after 7 days, BGM ^young treated with Ad-Dkk1 showed minimal activity (FIG. 23B). ^{Instead, BGM young} treated with Ad-Dkk1 showed widespread expression of the adipose-forming proteins PPAR-γ (FIGS. 23C, 23D) and Dlk1 (FIGS. 23E, 23F). Bone formation is as shown by micro-CT (FIG. 23G, FIG. 23H; quantified in FIG. 23I) and tissue morphometry analysis (FIG. 23J, 23K; quantified in FIG. 23L) for BGM. It was suppressed by the Ad-Dkk1 treatment. Therefore, the bone-forming ability of BGM depends on the endogenous Wnt signal.

Restoring the bone-forming ability by enhancing the endogenous Wnt signal in BGM ^aged FIGS. 23J-23L show that endogenous Wnt signaling can activate the bone-forming ability of BGM. .. We also investigated whether stimulation with Wnt was sufficient to enhance the effectiveness of BGM. BGM- ^aged was harvested, treated with L-WNT3A or liposome PBS (L-PBS), and then incubated at 37 ° C. (FIG. 24A). Absolute qRT-PCR analysis revealed a slight increase in Axin2 expression (Fig. 24B). Left1 increased slightly in response to L-WNT3A (FIG. 24B). Western analysis showed that both beta-catenin protein and Axin2 protein were elevated in response to L-WNT3A (Fig. 24C).

Mitotic activity within BGM was increased by L-WNT3A treatment. On the 4th day after transplantation ^{, cell proliferation was increased in BGM aged treated with L-WNT3A compared to BGM aged} treated with L-PBS (FIGS. 24D, 24E; quantified in FIG. 24F). There is). The effect on the cell cycle was transient: by 7 days post-transplant, BrdU uptake was comparable between L-PBS and L-WNT3A samples (FIGS. 24G, 24H; Quantified in FIG. 24I).

Cell differentiation within BGM ^{aged was assessed. In BGM aged} treated with L-WNT3A, the expression of the adipose-forming protein Dlk1 is low (FIG. 24J, FIG. 24K; quantified in FIG. 24L), and the expression of the bone-forming protein osteocalcin is It was high (FIG. 24M, FIG. 24N; quantified in FIG. 24O). ^{New bone formation was found only within BGM aged} treated with L-WNT3A (FIG. 24P, 24Q; quantified in FIG. 24R). Treatment with L-WNT3A did not affect engraftment efficiency (FIGS. 27A, B; quantified in FIG. 27C), but analysis of programmed cell death revealed that BGM ^{aged treated with L-WNT3A.} It was confirmed that the number of TUNEL ^{+ ve} cells possessed was less than that of the sample treated with L-PBS (FIGS. 27D, 27E; quantified in FIG. 27F). Reduction of apoptosis was also observed 7 days after transplantation (FIG. 27G, 27H; quantified in FIG. 27I). The new bone formation was used as a stimulus for osteoclast-mediated bone remodeling and TRAP activity in the vicinity of the newly formed osteoid matrix ^{in the L-WNT3A treated BGM-aged sample.} Was observed (FIG. 27J, 27K; quantified in FIG. 27L).

L-WNT3A ^{activates stem cells in BGM aged} and improves bone development in spinal fixation models. It is also a cell population in BGM and contributes to the increase in bone formation ability mediated by L-WNT3A. We also identified the population that became. Two stem cell populations were isolated from BGM using standard procedures and assessed based on their Wnt responsiveness using L-PBS (as a control) or L-WNT3A.

Analysis of the passage of time revealed the response of stem cells to L-WNT3A treatment. Within 15 hours of exposure to L-WNT3A, 4-fold activation of Axin2 was observed, and after 36 hours, maximal activation of Axin2 was achieved (FIG. 25A). The effect was transient, as indicated by the reduced expression of Axin2 in the stem cells at 60 hours later (FIG. 25A). A second stem cell population isolated from BGM was used to verify that stem cells responded to L-WNT3A (FIG. 25B).

The activated state of BGM was shown by qRT-PCR. BGM- ^aged was harvested, treated with L-WNT3A or L-PBS, and then analyzed for its Wnt responsive state using Axin2 expression and Left1 expression (FIG. 25C). Within 12 hours, an increase in Left1 became detectable. Within 24 hours, both Axin2 and Left1 were elevated (Fig. 25C). These analyzes confirm the WNT-mediated activation state of BGM, which is referred ^{to herein as BGM ACT.}

We also investigated the therapeutic potential of ^{BGM ACT} using a rat spinal fusion model. The cortex was removed from the transverse processes of the 4th and 5th lumbar vertebrae (eg, L4-5) (FIG. 25D), and during this procedure, autologous BGM ^aged from the iliac crest was harvested and L-WNT3A (L-WNT3A). Or L-PBS) for 1 hour. The resulting material, BGM ^ACT (or BGM ^aged ), was then implanted between the L4 and L5 protrusions, and between the L4 and L5 protrusions (FIG. 25E). On the second day after surgery, the volume of BGM was assessed by micro-CT. These analyzes ^{verified that BGM aged} (FIG. 25F) and BGM ^ACT (FIG. 25G) initially contained equivalent amounts of calcified tissue (see also FIG. 26).

49 days after surgery, the volume of new bone formation was reassessed. Three-dimensional reconstruction of micro-CT data ^{confirmed that bone regeneration within the BGM-aged} site was low (gray; FIG. 25H). In contrast, ^{sites treated with BGM ACT} showed evidence of robust bone formation and fusion of transverse processes (blue; FIG. 25I). New bone volumes were quantified between the L4 and L5 transverse processes and between the L4 and L5 transverse processes. Compared to BGM ^{aged , BGM ACT} resulted in a high degree of matrix calcification (Fig. 25J). Therefore, L-WNT3A treatment improves the bone-forming ability of autologous grafts derived from aged animals.

(Example 4)
Expression of Wnt3a from a Serum-Free CHO Cell-Specific Vector CHO Suspended (CHO-S) cells secreting Wnt3a are also cultured in a suitable expression medium, such as Invitogen's expression medium, under serum-free conditions. It could be. A CHO-S cell line compatible with cGMP and two vectors for cloning WNT3A were used. One vector is a TOPO® adaptive bicistronic plasmid that allows rapid cloning of genes containing secreted signals to mammals and genes of interest downstream of the CMV promoter. It was a plasmid. Dihydrofolate reductase selectable markers allow for rapid selection. This vector was used for transient transfection of CHO-S cells. Another vector was pTageT. This vector was used for transient transfection of CHO-S cells and was also used to create a stable cell line expressing WNT3A.

The conditioned medium (CM) derived from the CHO cell culture was collected and pooled between 3 and 13 days after induction. Based on an analysis of protein production in CM on each day, the number of days in this range was determined to be optimal under culture conditions. Under these conditions, it was observed that high protein production occurred between days 3 and 13, while cells began to die after day 13.

(Example 5)
Replacing Serum with Specified Substitutes To determine if they can be replaced with a defined set of lipids, examine the lipid components in the serum. Charcoal exfoliation removes non-polar lipophilic materials (viruses, growth factors, hormones) from the serum. CHO cells grown in charcoal-exfoliated FBS do not secrete Wnt3a as compared to control conditions in which CHO cells are grown in 10% FBS. These data support the conclusion that the lipophilic component of serum is required for the secretion of Wnt3a. Growth rate of CHO cells by supplementation with Lipid Mixture 1 (Sigma) containing cholesterol, tocopherol, and non-animal fatty acids (linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid) Is improved and Wnt3a secretion is restored to CHO cells.

(Example 6)
Gradual reduction of serum dependence We will also investigate sequential culture as a means to reach a serum-independent process for producing WNT3A. The growth rate of CHO cells is reduced at the same time as the reduction of serum, and as a result, it is important to shift the time frame for collecting the conditioned medium. Optionally, the cells are adapted to 0.5% FBS.

In the above description, the present invention has been described with reference to a specific embodiment, but the present invention is not limited in this way. In fact, from the above description, a variety of modifications of the present invention will be apparent to those skilled in the art in addition to the modifications set forth herein, which are within the scope of the appended claims. Will fit.

Claims (13)

A composition for the treatment of bone defects in a subject, comprising a mammalian Wnt 3a polypeptide and an aqueous solution containing liposomes.
The liposome contains a phospholipid which is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC), and the liposome contains cholesterol, and the concentration of DMPC and cholesterol is 70:30 to 90:10. Specified by the ratio between
The composition ex vivo contacts a sample containing mammalian bone marrow stem cells; and provides treatment for the bone defect when the composition is implanted at the site of the bone defect. A composition characterized by that. The composition according to claim 1, wherein the contact temperature is 0 ° C. to 37 ° C. or 20 ° C. to 25 ° C. The composition according to claim 1, wherein the contact time is 30 minutes to 4 hours. The composition according to claim 1, wherein the mammalian bone marrow cells include bone marrow stem cells and bone marrow progenitor cells. The composition according to claim 1, wherein the mammalian bone marrow cells are autologous bone marrow cells. The composition of claim 1, wherein the Wnt 3a polypeptide is a lipid modified at the amino acid position corresponding to the amino acid residue 209 set forth in SEQ ID NO: 1. The composition according to claim 6 , wherein the modification of the lipid is palmitoylation. The composition according to claim 6 , wherein the Wnt polypeptide is further glycosylated. The composition according to claim 1, which is a stable composition. The composition of claim 1, which is stable for up to about 106 days without substantial loss of activity. The composition according to claim 1, which is stable at a temperature between about 1 ° C. and about 8 ° C. The composition according to claim 1, which is stable under nitrogen. The composition according to claim 1, wherein the mammalian bone marrow cells are collected from a subject who is 35 years old or older.

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